U.S. patent application number 15/896428 was filed with the patent office on 2018-06-21 for covalent linkers in antibody-drug conjugates and methods of making and using the same.
The applicant listed for this patent is RC Biotechnologies, Inc.. Invention is credited to Jianmin Fang, Changjiang Huang, Hui Ye, Lezhi Zhang.
Application Number | 20180169250 15/896428 |
Document ID | / |
Family ID | 58052236 |
Filed Date | 2018-06-21 |
United States Patent
Application |
20180169250 |
Kind Code |
A1 |
Huang; Changjiang ; et
al. |
June 21, 2018 |
COVALENT LINKERS IN ANTIBODY-DRUG CONJUGATES AND METHODS OF MAKING
AND USING THE SAME
Abstract
The present invention provides novel and advantageous
compositions having a linker capable of covalently coupling one or
more free thiols of an antibody. Specifically, provided herein are
the molecular structures, synthetic pathways, coupling mechanisms,
and applications thereof as used in an antibody-drug conjugate
(ADC).
Inventors: |
Huang; Changjiang; (Los
Angeles, CA) ; Fang; Jianmin; (Palo Alto, CA)
; Ye; Hui; (Shandong, CN) ; Zhang; Lezhi;
(Shandong, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RC Biotechnologies, Inc. |
Fremont |
CA |
US |
|
|
Family ID: |
58052236 |
Appl. No.: |
15/896428 |
Filed: |
February 14, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2016/046987 |
Aug 15, 2016 |
|
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15896428 |
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62205121 |
Aug 14, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/545 20170801;
A61K 47/54 20170801; A61K 47/6803 20170801; A61P 35/00 20180101;
C07K 16/32 20130101; A61K 47/6811 20170801; A61K 47/6855 20170801;
C07K 16/2896 20130101; A61K 47/6869 20170801; A61K 47/6889
20170801 |
International
Class: |
A61K 47/54 20060101
A61K047/54; A61K 47/68 20060101 A61K047/68; C07K 16/28 20060101
C07K016/28; C07K 16/32 20060101 C07K016/32; A61P 35/00 20060101
A61P035/00 |
Claims
1. A linker-active agent comprising: ##STR00076## or a
pharmaceutically acceptable salt thereof, wherein: R.sup.1 and
R.sup.2 are functional groups that can react with thiols; L.sup.1,
L.sup.2, and L represent linker or can be null; M represents a
linking group; Q represents an active agent or can be null; m
represents an integer selected from the group consisting of 0, 1,
2, 3, 4, 5 and 6; n represents an integer selected from the group
consisting of 0, 1, 2, 3, 4, 5, 6, 7 and 8.
2. The linker-active agent or a pharmaceutically acceptable salt
thereof as claimed in claim 1, wherein R.sup.1 and R.sup.2 each
comprise at least one group selected from the group consisting of
maleimide, halogen, halogen-substituted functional groups, alkanal,
alkanone, sulfonyl-alkene, silane, isocyanate, and norbornene, and
wherein R.sup.1 and R.sup.2 cannot both be maleimide and R.sup.1
and R.sup.2 cannot both be halogen.
3. The linker-active agent or a pharmaceutically acceptable salt
thereof as claimed in claim 1, wherein R.sup.1 and R.sup.2 are
independently selected from the group consisting of: ##STR00077##
and derivatives thereof, wherein R.sup.5, R.sup.6, and R.sup.7 are
independently selected from the group consisting of H, halogen,
C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6 alkenyls, C.sub.2-C.sub.6
alkynyls, aromatics, heteroaryls, C.sub.3-C.sub.9 cycloalkyls,
C.sub.3-C.sub.9 heterocyclyl, polyethylene glycol, NO.sub.2, CN,
SCN, OR.sup.8, SR.sup.9, NR.sup.10R.sup.11, C(.dbd.O)R.sup.12,
C(.dbd.O)OR.sup.8, C(.dbd.O)NR.sup.10R.sup.11, C(.dbd.S)OR.sup.8,
C(.dbd.S)NR.sup.10R.sup.11, C(.dbd.S)SR.sup.9,
NR.sup.10(C.dbd.O)R.sup.12, NR.sup.10(C.dbd.S)NR.sup.10R.sup.11,
O(C.dbd.O)NR.sup.10R.sup.11, SO.sub.2R.sup.9,
S(.dbd.O).sub.2OR.sup.8, and combinations thereof, wherein R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12 are independently selected
from the group consisting of H, C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyls, C.sub.2-C.sub.6 alkynyls, and
combinations thereof, wherein X is selected from the group
consisting of F, Cl, Br, and I.
4. The linker-active agent or a pharmaceutically acceptable salt
thereof as claimed in claim 1, wherein R.sup.1 and R.sup.2 are
different.
5. The linker-active agent or a pharmaceutically acceptable salt
thereof as claimed in claim 1, wherein M is selected from the group
consisting of C--R.sup.5, N, B, P, Si--R.sup.5, aromatics,
heteroaryls, cycloalkyls, and (heterocyclyl)alkyls, and wherein
R.sup.5 is selected from the group consisting of H, C.sub.1-C.sub.6
alkyls, C.sub.2-C.sub.6 alkenyls, and C.sub.2-C.sub.6 alkynyls.
6. The linker-active agent or a pharmaceutically acceptable salt
thereof as claimed in claim 1, wherein L.sup.1 and L.sup.2 are
independently selected from the group consisting of C.sub.1-C.sub.6
alkyls, C.sub.2-C.sub.6 alkenyls, C.sub.2-C.sub.6 alkynyls,
aromatics, heteroaryls, C.sub.3-C.sub.9 cycloalkyls,
C.sub.3-C.sub.9 heterocyclyl, polyethylene glycol, O, S, NR.sup.6,
C(.dbd.O), C(.dbd.O)O, C(.dbd.O)NR.sup.6, C.dbd.NR.sup.6,
C(.dbd.S)O, C(.dbd.S)NR.sup.6, C(.dbd.S)S, NR.sup.6(C.dbd.O),
NR.sup.6(C.dbd.S)NR.sup.7, O(C.dbd.O)NR.sup.6, S(.dbd.O).sub.2, and
combinations thereof, and wherein R.sup.6 and R.sup.7 are
independently selected from the group consisting of H,
C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6 alkenyls, and
C.sub.2-C.sub.6 alkynyls.
7. The linker-active agent or a pharmaceutically acceptable salt
thereof as claimed in claim 1, wherein L comprises a non-cleavable
unit and/or a cleavable unit, or L comprises a C.sub.1-C.sub.9
alkyl, C.sub.2-C.sub.9alkenyl, C.sub.2-C.sub.9 alkynl, aromatic,
heteroaryl, C.sub.3-C.sub.9 cycloalkyl, C.sub.3-C.sub.9
heterocyclyl, polyethylene glycol, O, S, NR.sup.8, C(.dbd.O),
C(.dbd.O)O, C(.dbd.O)NR.sup.8, C.dbd.NR.sup.8, C(.dbd.S)O,
C(.dbd.S)NR.sup.8, C(.dbd.S)S, NR.sup.8(C.dbd.O),
NR.sup.8(C.dbd.S)NR.sup.9, O(C.dbd.O)NR.sup.8, S(.dbd.O).sub.2,
Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB,
Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB,
Ala-PAB, PAB, or a combination thereof, and wherein R.sup.8 and
R.sup.9 are independently selected from the group consisting of H,
C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6 alkenyls, and
C.sub.2-C.sub.6 alkynyls.
8. The linker-active agent or a pharmaceutically acceptable salt
thereof as claimed in claim 1, wherein Q is selected from the group
consisting of tubulin binders, DNA alkylators, DNA intercalators,
enzyme inhibitors, immune modulators, visualization agents,
peptides, and nucleotides, or Q is selected from the group
consisting of Maytansinoids, Auristatins, Calicheamicins,
Doxorubicins, Duocarmycins, and Pyrrolobenzodiazepines.
9. The linker-active agent or a pharmaceutically acceptable salt
thereof as claimed in claim 1, wherein the structure of Formula I
is any one selected from the group consisting of: ##STR00078##
##STR00079## ##STR00080##
10. An antibody-drug conjugate having the following formula:
##STR00081## or a pharmaceutically acceptable salt thereof,
Wherein: L.sup.1, L.sup.2, and L represent linker or can be null; M
represents a linking group; Q represents an active agent or can be
null; m represents an integer selected from the group consisting of
0, 1, 2, 3, 4, 5 and 6; n represents an integer selected from the
group consisting of 0, 1, 2, 3, 4, 5, 6, 7 and 8; A is a targeting
moiety; S is a sulfur atom of a thiol group of the targeting
moiety; and each of R.sup.3 and R.sup.4 is formed as the result of
a reaction between S and a functional group that can react with
thiols.
11. The antibody-drug conjugate as claimed in claim 10, wherein
R.sup.3 and R.sup.4 are different, and wherein each of R.sup.3 and
R.sup.4 is formed, independently, as the result of a reaction
between a thiol group and a group selected from the group
consisting of ##STR00082## and derivates thereof, wherein R.sup.5,
R.sup.6, and R.sup.7 are independently selected from the group
consisting of H, halogen, C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6
alkenyls, C.sub.2-C.sub.6 alkynyls, aromatics, heteroaryls,
C.sub.3-C.sub.9 cycloalkyls, C.sub.3-C.sub.9 heterocyclyl,
polyethylene glycol, NO.sub.2, CN, SCN, OR.sup.8, SR.sup.9,
NR.sup.10R.sup.11, C(.dbd.O)R.sup.12, C(.dbd.O)OR.sup.8,
C(.dbd.O)NR.sup.10R.sup.11, C(.dbd.S)OR.sup.8,
C(.dbd.S)NR.sup.10R.sup.11, C(.dbd.S)SR.sup.9,
NR.sup.10(C.dbd.O)R.sup.12, NR.sup.10(C.dbd.S)NR.sup.10R.sup.11,
O(C.dbd.O)NR.sup.10R.sup.11, SO.sub.2R.sup.9,
S(.dbd.O).sub.2OR.sup.8, and combinations thereof, wherein R.sup.8,
R.sup.9, R.sup.10, R.sup.11, and R.sup.12 are independently
selected from the group consisting of H, C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyls, C.sub.2-C.sub.6 alkynyls, and
combinations thereof, and wherein X is selected from the group
consisting of F, Cl, Br, and I, or each of R.sup.3 and R.sup.4 is
independently selected from the group consisting of: ##STR00083##
derivatives thereof.
12. The antibody-drug conjugate as claimed in claim 10, wherein M
is selected from the group consisting of C--R.sup.5, N, B, P,
Si--R.sup.5, aromatics, heteroaryls, cycloalkyls, and
(heterocyclyl)alkyls, and wherein R.sup.5 is selected from the
group consisting of H, C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6
alkenyls, and C.sub.2-C.sub.6 alkynyls.
13. The antibody-drug conjugate as claimed in claim 10, wherein
L.sup.1 and L.sup.2 are independently selected from the group
consisting of C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6 alkenyls,
C.sub.2-C.sub.6 alkynyls, aromatics, heteroaryls, C.sub.3-C.sub.9
cycloalkyls, C.sub.3-C.sub.9 heterocyclyl, polyethylene glycol, O,
S, NR.sup.6, C(.dbd.O), C(.dbd.O)O, C(.dbd.O)NR.sup.6,
C.dbd.NR.sup.6, C(.dbd.S)O, C(.dbd.S)NR.sup.6, C(.dbd.S)S,
NR.sup.6(C.dbd.O), NR.sup.6(C.dbd.S)NR.sup.7, O(C.dbd.O)NR.sup.6,
S(.dbd.O).sub.2, and combination thereof, and wherein R.sup.6 and
R.sup.7 are independently selected from the group consisting of H,
C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6 alkenyls, and
C.sub.2-C.sub.6 alkynyls.
14. The antibody-drug conjugate as claimed in claim 10, wherein L
comprises a non-cleavable unit and/or a cleavable unit, or L
comprises a C.sub.1-C.sub.9 alkyl, C.sub.2-C.sub.9 alkenyl,
C.sub.2-C.sub.9 alkynyl, aromatic, heteroaryl, C.sub.3-C.sub.9
cycloalkyl, C.sub.3-C.sub.9 heterocyclyl, polyethylene glycol, O,
S, NR.sup.8, C(.dbd.O), C(.dbd.O)O, C(.dbd.O)NR.sup.8,
C.dbd.NR.sup.8, C(.dbd.S)O, C(.dbd.S)NR.sup.8, C(.dbd.S)S,
NR.sup.8(C.dbd.O), NR.sup.8(C.dbd.S)NR.sup.9, O(C.dbd.O)NR.sup.9,
S(.dbd.O).sub.2, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB,
Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg,
Ala-Ala-Asn-PAB, Ala-PAB, PAB, or a combination thereof, and
wherein R.sup.8 and R.sup.9 are independently selected from the
group consisting of H, C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6
alkenyls, and C.sub.2-C.sub.6 alkynyls.
15. The antibody-drug conjugate as claimed in claim 10, wherein the
structure of Formula II is any one selected from the group
consisting of: ##STR00084## ##STR00085## ##STR00086## or a
pharmaceutically acceptable salt thereof.
16. The antibody-drug conjugate as claimed in claim 10, wherein A
comprises a monoclonal antibody, an antibody fragment, an antibody
surrogate, an antibody variant, a protein ligand, a protein
scaffold, a peptide, or a small molecule ligand.
17. A method for treating a pathological condition in a subject
wherein said method comprises administering, to a subject in need
of such treatment, an antibody-drug conjugate of claim 10.
18. The method, according to claim 17, wherein said pathological
condition is selected from the group consisting of cancer,
inflammatory disorders, autoimmune disorders, disorders of the
nervous system, and cardiovascular disorders.
19. The method, according to claim 17, wherein the cancer is
selected from the group consisting of carcinoma, lymphoma,
blastoma, sarcoma, and leukemia or lymphoid malignancies, or a HER2
positive cancer, or breast cancer.
20. The method, according to claim 17, wherein A is an antibody, or
a fragment thereof, that binds to HER2, and Q is a cytotoxin.
21. A diagnostic method that comprises contacting a sample,
suspected of having an analyte of interest, with the antibody-drug
conjugate of claim 10, wherein the target moiety selectively binds
to the analyte of interest and Q is a detectable entity and/or is
an entity to which one or more additional entities will bind
wherein said one or more additional entities are, themselves,
detectable entities, and wherein said method further comprises
analyzing the sample to determine the presence of a detectable
entity, which presence would be indicative of the presence of the
analyte of interest.
22. The diagnostic method, according to claim 21, wherein the
analyte is associated with cancer or the analyte is HER2.
23. The diagnostic method, according to claim 21, wherein A is an
antibody or an antibody fragment.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation-in-part
application under 35 USC 111(a) of PCT international application
No. PCT/US2016/046987, filed on Aug. 15, 2016, which claims the
benefit of U.S. Provisional Application Ser. No. 62/205,121, filed
Aug. 14, 2015, all of which are hereby incorporated by reference
herein in their entirety.
BACKGROUND OF THE INVENTION
[0002] Antibody drug conjugates (ADCs), a rapidly growing class of
targeted therapeutics, represent a promising new approach toward
improving both the selectivity and the cytotoxic activity of cancer
drugs. An example of ADC drug having been approved for therapeutic
use in the U.S. is brentuximab vedotin (ADCETRIS.RTM.), a chimeric
anti-CD30 antibody conjugated to monomethyl auristatin E used in
treating anaplastic large cell lymphoma and Hodgkin lymphoma.
[0003] One conventional method employed in the design of
antibody-drug conjugates (ADC) involves the coupling of drug
molecules to thiol groups of antibody chains via a linking moiety.
Free thiol groups are obtained as a result of breaking the cysteine
interchain disulfide bonds of an antibody via a reduction reaction.
A typical antibody contains 4 interchain disulfide bonds (2 between
the heavy chains and 2 between the heavy and light chains). These
interchain disulfides can be selectively reduced with
dithiothreitol, tris(2-carboxyethyl)phosphine, or other mild
reducing agents, to result in 8 reactive sulfhydryl groups for
conjugation. This method can link up to eight drug molecules to a
given antibody.
[0004] Due to the fact that at least two disulfide bonds are
broken, ADCs designed using this principle are unstable once
entered into circulation, and thus the half life of ADCs will be
shortened. As a result, recent development in ADC design and
synthesis adopts a different approach, namely, one that relies on
covalently connecting two thiol groups by a coupling agent, thereby
establishing thiol bridges between the two heavy chains and between
the heavy and the light chains of a given antibody. Current
research efforts exploring such an approach mainly focus on
designing the structure of a coupling agent that not only has the
functionalities to bridge two thiol groups covalently, but also
encompasses the necessary components to facilitate specific
biological activities.
[0005] Earlier studies in the field have utilized bis-maleimides to
react with the two thiol groups resulted from a broken disulfide
bond. The covalent coupling between maleimides and thiols is a
classic alkene conversion reaction. More recently studied
thiol-bridging reactions are also based upon this principle, with
exemplary reactions involving maleimides, bis-maleimides, and
maleimides with halogen substituents. To date, however,
thiol-bridging linker compositions are limited to maleimides-based
compounds only, and oftentimes not specified for applications in
tumor-targeting ADCs. For example, previously disclosed methods of
covalent thiol-coupling involving the use of similar
maleimides-based compounds did not specify their applications in
coupling with active agents such as tumor-targeting drug molecules,
proteins, or polypeptides (see, for example, PCT Patent Application
Publication No. WO 2013132268). Other methods that specifically
disclosed applications in tumor-targeting ADCs utilizing
maleimides-based compounds did not employ the covalent
thiol-bridging mechanism in which one linker simultaneously reacts
with two thiol groups, as provided by the present invention (see,
for example, Chinese Patent Application Publication No. CN
103933575).
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention provides novel and advantageous
linkers for coupling an antibody to another compound. In addition
to the linkers which the structural synthesis and the use thereof
described herein, the subject invention also provides
antibody/active-agent conjugates, and applications thereof as used
in, for example, antibody-drug conjugate applications.
[0007] Some embodiments provide a linker-active agent having the
structure of Formula I:
##STR00001##
or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 and
R.sup.2 are functional groups that can react with thiols; L.sup.1,
L.sup.2, and L represent linker or can be null; M represents a
linking group; Q represents an active agent or can be null; m
represents an integer selected from the group consisting of 0, 1,
2, 3, 4, 5 and 6; n represents an integer selected from the group
consisting of 0, 1, 2, 3, 4, 5, 6, 7 and 8. In some embodiments,
R.sup.1 and R.sup.2 are independently selected from the group
consisting of
##STR00002##
and derivatives thereof;
[0008] Some embodiments provide a targeting moiety and a
linker-active agent conjugate having the structure of Formula
II:
##STR00003##
or a pharmaceutically acceptable salt thereof,
[0009] A is a targeting moiety;
[0010] L.sup.1, L.sup.2, L represent linker or null;
[0011] M is a linking group;
[0012] Q is an active agent or null;
[0013] m is an integer selected from the group consisting of 0, 1,
2, 3, 4, 5 and 6;
[0014] n is an integer selected the group consisting of 0, 1, 2, 3,
4, 5, 6, 7 and 8;
[0015] Each of R.sup.3 and R.sup.4 is independently selected from
the group consisting of:
##STR00004##
derivatives thereof, or is null.
[0016] As used herein, reference to "null" means that the entity is
not present.
BRIEF DESCRIPTION OF DRAWINGS
[0017] FIG. 1 shows a schematic of covalent thiol-bridge
established by a linker reacting with two neighboring thiol groups
of a broken or reduced disulfide bond of antibody.
[0018] FIG. 2 is an SDS-PAGE image of an exemplary anti-Her-2 ADC
comprising an anti-Her-2 monoclonal antibody conjugated with a
cytotoxin prepared using Mc-VC-PAB-MMAE.
[0019] FIG. 3 shows an HIC-HPLC chromatogram of MSL-C31.
[0020] FIG. 4 shows the results of reduced SDS-PAGE of MSL-C75.
[0021] FIG. 5 shows a HIC-HPLC chromatogram of MSL-C75.
[0022] FIG. 6 shows the result of reduced SDS-PAGE for, a) naked
antibody, b) CD59-C78.
[0023] FIG. 7 shows an HIC-HPLC chromatogram of CD59-C78.
[0024] FIG. 8 shows the change in body weight of BALB/c nude mice
after administration of ADCs of the present invention.
[0025] FIG. 9 shows the N87 tumor volume of BALB/c nude mice for
ADCs of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0026] In the following detailed description, reference is made to
the accompanying drawings, depicting exemplary, non-limiting and
non-exhaustive embodiments of the invention. These embodiments are
described in sufficient detail to enable those having skill in the
art to practice the invention, and it is understood that other
embodiments may be used, and other changes may be made, without
departing from the spirit or scope of the invention. The following
detailed description is, therefore, not to be taken in a limiting
sense, and the scope of the invention is defined only by the
appended claims. All patents, patent applications, provisional
applications, and publications referred to or cited herein are
incorporated by reference in their entirety, including all figures
and tables, to the extent that they are not inconsistent with the
explicit teachings of this specification.
[0027] The present invention provides novel and advantageous Linker
molecules capable of covalently coupling to two free thiols of an
antibody via one terminus of the Linker, and attaching to an active
agent at another terminus of the Linker. Antibody/active-agent
conjugates herein are also provided, including, for example,
antibody-drug conjugates (ADCs). In some embodiments, ADCs provided
herein incorporate an anti-cancer drug as the active agent.
Definitions
[0028] As used herein, abbreviations of common organic compounds
are defined below:
BOC tert-Butoxycarbonyl
Fmoc 9-Fluorenylmethoxycarbonyl
[0029] Temperature in degrees Centigrade
DIPEA Diisopropylethylamine
[0030] DMF N,N'-Dimethyl formamide R.T. Room temperature
EtOH Ethanol
h Hour
Et.sub.3N Triethylamine
HOBt N-Hydroxybenzotriazole
[0031] Prep-HPLC Preparative high performance liquid
chromatography
NaHCO.sub.3 Sodium Bicarbonate
[0032] DCC Dicyclohexyl carbodiimide
MeCN Acetonitrile
[0033] EDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide DIC
N,N'-diisopropyl carbodiimide
DEA Diethylamine
[0034] K.sub.2CO.sub.3 Potassium carbonate
M Mole/liter
mL Milliliter
[0035] MgSO.sub.4 Magnesium sulphate
THF Tetrahydrofuran
[0036] CH.sub.2Cl.sub.2 Dichloromethane NaCl Sodium chloride
NaSO.sub.4 Sodium sulfate
HCl Hydrochloric Acid
[0037] LC-MS Liquid chromatography-mass spectrometry
CHCl.sub.3 Trichloromethane
[0038] NaAc Sodium acetate Ac.sub.2O Acetic anhydride TFA
Trifluoroacetic acid NaOH Sodium hydroxide
[0039] The term "active agent" as used according to the present
invention includes any natural or synthetic substance that has a
physiological effect when administered to an animal. The active
agent can be utilized in accordance with the invention to treat,
for example, warm blooded animals, particularly mammals including
humans, veterinarian animals and farm animals. The active agent may
act on, or be visualized at, a desired target within, or on, the
animal body, including tumor tissue.
[0040] Non-limiting examples of "active agents" are drugs acting at
synaptic sites and neuroeffector junctional sites; general and
local analgesics; hypnotics and sedatives; drugs for the treatment
of psychiatric disorders such as depression and schizophrenia;
anti-epileptics and anticonvulsants; drugs for the treatment of
Parkinson's and Huntington's disease, aging and Alzheimer's
disease; excitatory amino acid antagonists, neurotrophic factors
and neuroregenerative agents; trophic factors; drugs aimed at the
treatment of central nervous system (CNS) trauma or stroke; drugs
for the treatment of addiction and drug abuse; drugs for the
treatment of bacterial, viral and/or microbial infections, such as
influenza, HIV, herpes, chicken pox, and the like; antacoids and
anti-inflammatory drugs; chemotherapeutic agents for parasitic
infections and diseases caused by microbes; immunosuppressive
agents; anti-cancer drugs; hormones and hormone antagonists; heavy
metals and heavy metal antagonists; antagonists for non-metallic
toxic agents; cytostatic agents; visualization agents and other
diagnostic substances; immunoactive and immunoreactive agents;
transmitters and their respective receptor agonists and receptor
antagonists, their respective precursors and metabolites;
transporter inhibitors; antibiotics; antispasmodics;
antihistamines; antinauseants; relaxants; stimulants; sense and
antisense oligonucleotides; cerebral dilators; psychotropics;
antimanics; vascular dilators and constrictors; anti-hypertensives;
drugs for migraine treatment; hyperglycemic and hypoglycemic
agents; minerals and nutritional agents; anti-obesity drugs;
anabolics; anti-asthmatics; and mixtures thereof.
[0041] An "antibody," also known as an immunoglobulin, is a large
Y-shaped protein used by the immune system to identify and
neutralize foreign entities such as bacteria and viruses. An
antibody has four polypeptide chains, two identical heavy chains
and two identical light chains connected by cysteine disulfide
bonds.
[0042] A "monoclonal antibody" is a monospecific antibody where all
the antibody molecules are identical because they are made by
identical immune cells that are all clones of a unique parent cell.
Monoclonal antibodies can be prepared by fusing myeloma cells with
the spleen cells from a mouse (or B-cells from a rabbit) that has
been immunized with the desired antigen, then purifying the
resulting hybridomas by such techniques as affinity purification.
Recombinant monoclonal antibodies can be prepared in viruses or
yeast cells rather than in mice, through technologies including
repertoire cloning or phage display/yeast display, the cloning of
immunoglobulin gene segments to create libraries of antibodies with
slightly different amino acid sequences from which antibodies with
desired specificities may be obtained. The resulting antibodies may
be prepared on a large scale by fermentation.
[0043] "Chimeric" or "humanized" antibodies are antibodies
containing a combination of the original (usually mouse) and human
DNA sequences used in the recombinant process, such as those in
which mouse DNA encoding the binding portion of a monoclonal
antibody is merged with human antibody-producing DNA to yield a
partially-mouse, partially-human monoclonal antibody.
Full-humanized antibodies are produced using transgenic mice
(engineered to produce human antibodies) or phage display
libraries.
[0044] A "linker" is a moiety with two reactive termini, one for
binding, or otherwise associating with, a biological moiety or a
fragment thereof, such as an antibody (or fragment thereof), and
the other for conjugation to an active agent such as a
cytotoxin.
[0045] A "cytotoxin" is an entity that, when in the presence of a
cell, such as a cancer cell, is toxic or induces key functional
changes to that cell.
[0046] As used herein, "alkyl" refers to a straight or branched
hydrocarbon chain that is fully saturated (i.e., contains no double
or triple bonds). The alkyl group may have 1 to 9 carbon atoms
(whenever it appears herein, a numerical range such as "1 to 9"
refers to each integer in the given range; e.g., "1 to 9 carbon
atoms" means that the alkyl group may comprise 1 carbon atom, 2
carbon atoms, 3 carbon atoms, etc., up to and including 9 carbon
atoms, although the present definition also covers the occurrence
of the term "alkyl" where no numerical range is designated). The
alkyl group may also be a medium size alkyl having 1 to 9 carbon
atoms. Typical alkyl groups include, but are in no way limited to,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl,
pentyl, hexyl, and the like.
[0047] As used herein, "alkenyl" refers to a straight or branched
hydrocarbon chain containing one or more double bonds. The alkenyl
group may have 2 to 9 carbon atoms, although the present definition
also covers the occurrence of the term "alkenyl" where no numerical
range is designated. The alkenyl group may also be a medium size
alkenyl having 2 to 9 carbon atoms. The alkenyl group could also be
a lower alkenyl having 2 to 4 carbon atoms. The alkenyl group may
be designated as "C.sub.2-4 alkenyl" or similar designations. By
way of example only, "C.sub.2-4 alkenyl" indicates that there are
two to four carbon atoms in the alkenyl chain, i.e., the alkenyl
chain is selected from the group consisting of ethenyl,
propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl,
buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl,
1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl,
buta-1,2,-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groups
include, but are in no way limited to, ethenyl, propenyl, butenyl,
pentenyl, and hexenyl, and the like.
[0048] As used herein, "alkynyl" refers to a straight or branched
hydrocarbon chain comprising one or more triple bonds. The alkynyl
group may have 2 to 9 carbon atoms, although the present definition
also covers the occurrence of the term "alkynyl" where no numerical
range is designated. The alkynyl group may also be a medium size
alkynyl having 2 to 9 carbon atoms. The alkynyl group could also be
a lower alkynyl having 2 to 4 carbon atoms. The alkynyl group may
be designated as "C.sub.2 4 alkynyl" or similar designations. By
way of example only, "C.sub.2-4 alkynyl" indicates that there are
two to four carbon atoms in the alkynyl chain, i.e., the alkynyl
chain is selected from the group consisting of ethynyl,
propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and
2-butynyl. Typical alkynyl groups include, but are in no way
limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and
the like.
[0049] The term "aromatic" refers to a ring or ring system having a
conjugated .pi. electron system and includes both carbocyclic
aromatic (e.g., phenyl) and heterocyclic aromatic groups (e.g.,
pyridine). The term includes monocyclic or fused-ring polycyclic
(i.e., rings which share adjacent pairs of atoms) groups provided
that the entire ring system is aromatic.
[0050] As used herein, "cycloalkyl" means a fully saturated
carbocyclyl ring or ring system. Examples include, but are not
limited to, cyclopropyl, cyclobutyl, cyclopentyl, and
cyclohexyl.
[0051] As used herein, "heteroaryl" refers to an aromatic ring or
ring system (i.e., two or more fused rings that share two adjacent
atoms) that comprise(s) one or more heteroatoms, that is, an
element other than carbon, including but not limited to, nitrogen,
oxygen and sulfur, in the ring backbone. When the heteroaryl is a
ring system, every ring in the system is aromatic. The heteroaryl
group may have 5-18 ring members (i.e., the number of atoms making
up the ring backbone, including carbon atoms and heteroatoms),
although the present definition also covers the occurrence of the
term "heteroaryl" where no numerical range is designated. Examples
of heteroaryl rings include, but are not limited to, furyl,
thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl,
pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl,
pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,
quinolinyl, isoquinlinyl, benzimidazolyl, benzoxazolyl,
benzothiazolyl, indolyl, isoindolyl, and benzothienyl.
[0052] As used herein, "heterocyclyl" means a non-aromatic cyclic
ring or ring system containing at least one heteroatom in the ring
backbone. Heterocyclyls may be joined together in a fused, bridged
or spiro-connected fashion. Heterocyclyls may have any degree of
saturation provided that at least one ring in the ring system is
not aromatic. The heteroatom(s) may be present in either a
non-aromatic or aromatic ring in the ring system. The heterocyclyl
group may have 3 to 20 ring members (i.e., the number of atoms
making up the ring backbone, including carbon atoms and
heteroatoms), although the present definition also covers the
occurrence of the term "heterocyclyl" where no numerical range is
designated. The heterocyclyl group may also be a medium size
heterocyclyl having 3 to 10 ring members. The heterocyclyl group
could also be a heterocyclyl having 3 to 6 ring members. Examples
of heterocyclyl rings include, but are not limited to, azepinyl,
acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl,
imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl,
piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl,
pyrrolidonyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl,
pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl,
1,4-dioxanyl, 1,3-oxathianyl, 1,4-oxathiinyl, 1,4-oxathianyl,
2H-1,2-oxazinyl, trioxanyl, hexahydro-1,3,5-triazinyl,
1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl, 1,3-dithiolanyl,
isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl,
oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl,
indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl,
tetrahydro-1,4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl,
benzimidazolidinyl, and tetrahydroquinoline.
[0053] A "(heterocyclyl)alkyl" is a heterocyclyl group connected,
as a substituent, via an alkylene group. Examples include, but are
not limited to, imidazolinylmethyl and indolinylethyl.
Linker/Active-Agent
[0054] In one aspect, the present invention provides a
linker-active agent that can be represented as Formula I:
##STR00005##
or a pharmaceutically acceptable salt thereof,
[0055] wherein:
[0056] L.sup.1, L.sup.2, and L represent a linker or null;
[0057] M is a linking group;
[0058] Q is an active agent or null;
[0059] m is an integer selected from the group consisting of 0, 1,
2, 3, 4, 5 and 6;
[0060] n is an integer selected from the group consisting of 0, 1,
2, 3, 4, 5, 6, 7 and 8;
[0061] each of R.sup.1 and R.sup.2 is a functional group that can
react with a thiol.
[0062] In some embodiments, each of the two thiol-reactive
functional groups may, independently, comprise a maleimide,
halogen, halogen-substituted functional groups, alkanal, alkanone,
sulfonyl-alkene, silane, isocyanate, or norbornene, but both cannot
be maleimide and both cannot be halogen.
[0063] In specific embodiments, each of R.sup.1 and R.sup.2 is
independently selected from the group consisting of:
##STR00006##
and derivatives thereof; [0064] wherein, in this embodiment,
R.sup.5, R.sup.6, and R.sup.7 are independently selected from the
group consisting of H, halogen, C.sub.1-C.sub.6 alkyls,
C.sub.2-C.sub.6 alkenyls, C.sub.2-C.sub.6 alkynyls, aromatics,
heteroaryls, C.sub.3-C.sub.9 cycloalkyls, C.sub.3-C.sub.9
heterocyclyl, polyethylene glycol, NO.sub.2, CN, SCN, OR.sup.8,
SR.sup.9, NR.sup.10R.sup.11, C(.dbd.O)R.sup.12, C(.dbd.O)OR.sup.8,
C(.dbd.O)NR.sup.10R.sup.11, C(.dbd.S)OR.sup.8,
C(.dbd.S)NR.sup.10R.sup.11, C(.dbd.S)SR.sup.9,
NR.sup.10(C.dbd.O)R.sup.12, NR.sup.10(C.dbd.S)NR.sup.10R.sup.11,
O(C.dbd.O)NR.sup.10R.sup.11, SO.sub.2R.sup.9,
S(.dbd.O).sub.2OR.sup.8, and combinations thereof, wherein R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12 are independently selected
from the group consisting of H, C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyls, C.sub.2-C.sub.6 alkynyls, and
combinations thereof. [0065] X is a halogen; in some embodiments X
can be F, Cl, Br, or I; [0066] R.sup.1 and R.sup.2 can be the same
or different.
[0067] In some embodiments, M is selected from the group consisting
of, for example, C--R.sup.5, N, B, P, Si--R.sup.5, aromatics,
heteroaryls, cycloalkyls, and (heterocyclyl)alkyls, wherein R.sup.5
is selected from the group consisting of H, C.sub.1-C.sub.6 alkyls,
C.sub.2-C.sub.6 alkenyls, and C.sub.2-C.sub.6 alkynyls.
[0068] In other embodiments, M is selected from the group
consisting of
##STR00007## ##STR00008##
[0069] In certain embodiments, M is selected from the group
consisting of
##STR00009##
[0070] In some embodiments, Q is an active agent, selected from the
group consisting of tubulin binders, DNA alkylators, DNA
intercalator, enzyme inhibitors, immune modulators, peptides, and
nucleotides.
[0071] In some embodiments, Q is selected from the group consisting
of Maytansinoids, Auristatins, Calicheamicins, Doxorubicins,
Duocarmycins, and Pyrrolobenzodiazepines.
[0072] In certain embodiments, Q is selected from the group
consisting of MMAE, MMAF, PBD dimer, DM1, and DM4. The active agent
may be, for example, any of those that are disclosed in WO
2013085925A1, which is incorporated herein, in its entirety, by
reference. "Drug" and "active agent" are used interchangeably
herein.
[0073] In some embodiments, each of the internal linking moieties
L.sup.1, L.sup.2 is independently selected from the group
consisting of C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6 alkenyls,
C.sub.2-C.sub.6 alkynyls, aromatics, heteroaryls, C.sub.3-C.sub.9
cycloalkyls, C.sub.3-C.sub.9 heterocyclyl, polyethylene glycol, O,
S, NR.sup.6, C(.dbd.O), C(.dbd.O)O, C(.dbd.O)NR.sup.6,
C.dbd.NR.sup.6, C(.dbd.S)O, C(.dbd.S)NR.sup.6, C(.dbd.S)S,
NR.sup.6(C.dbd.O), NR.sup.6(C.dbd.S)NR.sup.7, O(C.dbd.O)NR.sup.6,
S(.dbd.O).sub.2 and any combination thereof;
[0074] wherein, in this embodiment, R.sup.6 and R.sup.7 are
independently selected from the group consisting of H,
C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6 alkenyls, and
C.sub.2-C.sub.6 alkynyls.
[0075] In other embodiments, each of L.sup.1, L.sup.2 is
independently selected from the group consisting of:
##STR00010##
or is null.
[0076] In certain embodiments, each of L.sup.1, L.sup.2 is
independently selected from the group consisting of:
##STR00011##
or is null.
[0077] L represents a linker, connecting active agent Q to linking
group M. In some embodiments, L includes a non-cleavable unit, in
other embodiments, L includes a cleavable unit. The
linker/active-agent conjugate may have one linker, two linkers,
three linkers, four linkers, five linkers or six linkers.
[0078] In some embodiments, L is independently selected from the
group consisting of C.sub.1-C.sub.9 alkyls, C.sub.2-C.sub.9
alkenyls, C.sub.2-C.sub.9 alkynyls, aromatics, heteroaryls,
C.sub.3-C.sub.9 cycloalkyls, C.sub.3-C.sub.9 heterocyclyl,
polyethylene glycol, O, S, NR.sup.8, C(.dbd.O), C(.dbd.O)O,
C(.dbd.O)NR.sup.8, C.dbd.NR.sup.8, C(.dbd.S)O, C(.dbd.S)NR.sup.8,
C(.dbd.S)S, NR.sup.8(C.dbd.O), NR.sup.8(C.dbd.S)NR.sup.9,
O(C.dbd.O)NR.sup.8, S(.dbd.O).sub.2, Val-Cit-PAB, Val-Ala-PAB,
Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys,
Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB, and any combination
thereof.
[0079] In this embodiment, R.sup.8 and R.sup.9 are independently
selected from the group consisting of H, C.sub.1-C.sub.6 alkyls,
C.sub.2-C.sub.6 alkenyls, and C.sub.2-C.sub.6 alkynyls.
[0080] In some embodiments, L is null. When L is null, active agent
Q is connected directly to M.
[0081] In some embodiments, L is independently selected from the
group consisting of:
##STR00012## ##STR00013## ##STR00014##
or is null.
[0082] In some embodiments, L is independently selected from the
groups as followed:
##STR00015## ##STR00016##
or is null.
[0083] In some embodiments, the linker-active agent conjugate has
the following structure of Formula I:
##STR00017##
[0084] In some embodiments, the linker-active agent conjugate has
the following structure of Formula I:
##STR00018##
[0085] In some embodiments, the linker-active agent conjugate has
the following structure of Formula I:
##STR00019##
[0086] In some embodiments, the linker-active agent conjugate has
the following structure of Formula I:
##STR00020##
[0087] In some embodiments, the linker-active agent conjugate has
the following structure of Formula I:
##STR00021##
[0088] In some embodiments, the linker-active agent conjugate has
the following structure of Formula I:
##STR00022##
[0089] In some embodiments, the linker-active agent conjugate has
the following structure of Formula I:
##STR00023##
[0090] In some embodiments, the linker-active agent conjugate has
the following structure of Formula I:
##STR00024##
[0091] In some embodiments, the linker-active agent conjugate has
the following structure of Formula I:
##STR00025##
[0092] In some embodiments, the linker-active agent conjugate has
the following structure of Formula I:
##STR00026##
[0093] In some embodiments, the linker-active agent conjugate has
the following structure of Formula I:
##STR00027##
[0094] In some embodiments, the linker-active agent conjugate has
the following structure of Formula I:
##STR00028##
[0095] In another aspect, the present invention provides an
antibody drug conjugate having the following formula II:
##STR00029##
or a pharmaceutically acceptable salt thereof,
[0096] A is a targeting moiety;
[0097] S is a sulfur atom of a thiol group of the targeting
moiety;
[0098] L.sup.1, L.sup.2, L represent a linker or null;
[0099] M is a linking group;
[0100] Q is an active agent or null;
[0101] m is an integer selected from the group consisting of 0, 1,
2, 3, 4, 5 and 6;
[0102] n is an integer selected from the group consisting of 0, 1,
2, 3, 4, 5, 6, 7 and 8; and
[0103] each of R.sup.3 and R.sup.4 is formed as the result of a
reaction between S and a functional group that can react with
thiols.
[0104] In some embodiments, the functional groups that react with a
thiol may, independently, comprise a maleimide, halogen,
halogen-substituted functional groups, alkanal, alkanone,
sulfonyl-alkene, silane, isocyanate, or norbornene, but both cannot
be maleimide and both cannot be halogen.
[0105] In certain embodiments, each of R.sup.3 and R.sup.4 are
formed, independently, as the result of a reaction between a thiol
group and a group selected from the group consisting of
##STR00030##
and derivates thereof. [0106] wherein, in this embodiment, R.sup.5,
R.sup.6, and R.sup.7 are independently selected from the group
consisting of H, halogen, C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6
alkenyls, C.sub.2-C.sub.6 alkynyls, aromatics, heteroaryls,
C.sub.3-C.sub.9 cycloalkyls, C.sub.3-C.sub.9 heterocyclyl,
polyethylene glycol, NO.sub.2, CN, SCN, OR.sup.8, SR.sup.9,
NR.sup.10R.sup.11, C(.dbd.O)R.sup.12, C(.dbd.O)OR.sup.8,
C(.dbd.O)NR.sup.10R.sup.11, C(.dbd.S)OR.sup.8,
C(.dbd.S)NR.sup.10R.sup.11, C(.dbd.S)SR.sup.9,
NR.sup.10(C.dbd.O)R.sup.12, NR.sup.10(C.dbd.S)NR.sup.10R.sup.11,
O(C.dbd.O)NR.sup.10R.sup.11, SO.sub.2R.sup.9,
S(.dbd.O).sub.2OR.sup.8, and combinations thereof, wherein R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12 are independently selected
from the group consisting of H, C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyls, C.sub.2-C.sub.6 alkynyls, and
combinations thereof.
[0107] In certain embodiments R.sup.3 and R.sup.4 have 10 or fewer,
preferably 5 or fewer, carbon atoms, are cylic and/or comprise a
ketone group.
[0108] In specific embodiments, each of R.sup.3 and R.sup.4 is
independently selected from the group consisting of:
##STR00031##
and derivatives thereof. In one embodiment, R.sup.3 and R.sup.4 are
able to link 2 sulfhydryl groups and make light-heavy chains of an
antibody more stable.
[0109] When R.sup.3 or R.sup.4 is null, L.sup.1 or L.sup.2 is
conjugated directly to the targeting moiety.
[0110] R.sup.3 and R.sup.4 can be the same or different.
[0111] Only R.sup.3 is
##STR00032##
R.sup.3 and R.sup.4 can be the same.
[0112] In some embodiments, M is selected from the group consisting
of, for example, C--R.sup.5, C, N, B, P, Si--R.sup.5, aromatics,
heteroaryls, cycloalkyls, and (heterocyclyl)alkyls, R.sup.5 is
selected from the group consisting of H, C.sub.1-C.sub.6 alkyls,
C.sub.2-C.sub.6 alkenyls, and C.sub.2-C.sub.6 alkynyls. In other
embodiments, M is selected from the group consisting of
##STR00033##
[0113] In certain embodiments, M is selected from the group
consisting of
##STR00034##
[0114] In some embodiments, Q is an active agent selected from the
group consisting of tubulin binders, DNA alkylators, DNA
intercalator, enzyme inhibitors, immune modulators, peptides, and
nucleotides. "Drug" and "active agent" are used interchangeably
herein.
[0115] In some embodiments, Q is selected from the group consisting
of Maytansinoids, Auristatins, Calicheamicins, Doxorubicins,
Duocarmycins, and PBDs.
[0116] In certain embodiments, Q is selected from the group
consisting of MMAE, MMAF, PBD, DM1, and DM4. The active agent may
be, for example, any of those that are disclosed in WO
2013085925A1, which is incorporated herein, it its entirety, by
reference.
[0117] In some embodiments, each of the internal linking moieties
L.sup.1, L.sup.2 is independently selected from the group
consisting of C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6 alkenyls,
C.sub.2-C.sub.6 alkynyls, aromatics, heteroaryls, C.sub.3-C.sub.9
cycloalkyls, C.sub.3-C.sub.9 heterocyclyl, polyethylene glycol, O,
S, NR.sup.6, C(.dbd.O), C(.dbd.O)O, C(.dbd.O)NR.sup.6,
C.dbd.NR.sup.6, C(.dbd.S)O, C(.dbd.S)NR.sup.6, C(.dbd.S)S,
NR.sup.6(C.dbd.O), NR.sup.6(C.dbd.S)NR.sup.7, O(C.dbd.O)NR.sup.6,
S(.dbd.O).sub.2 and any combination thereof.
[0118] Wherein, in this embodiment, R.sup.6 and R.sup.7 are
independently selected from the group consisting of H,
C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6 alkenyls, and
C.sub.2-C.sub.6 alkynyls.
[0119] In other embodiments, each of L.sup.1 and L.sup.2 is
independently selected from the group consisting of:
##STR00035##
or is null.
[0120] In certain embodiments, each of L.sup.1 and L.sup.2 is
independently selected from the group consisting of:
##STR00036##
or is null.
[0121] L represents a linker, connecting drug Q to linking group M.
In some embodiments, L includes a non-cleavable unit, in other
embodiments, L includes a cleavable unit. The antibody drug
conjugate may have one linker, two linkers, three linkers, four
linkers, five linkers or six linkers.
[0122] In some embodiments, L is independently selected from the
group consisting of C.sub.1-C.sub.9 alkyls, C.sub.2-C.sub.9
alkenyls, C.sub.2-C.sub.9 alkynyls, aromatics, heteroaryls,
C.sub.3-C.sub.9 cycloalkyls, C.sub.3-C.sub.9 heterocyclyl,
polyethylene glycol, O, S, NR.sup.8, C(.dbd.O), C(.dbd.O)O,
C(.dbd.O)NR.sup.8, C.dbd.NR.sup.8, C(.dbd.S)O, C(.dbd.S)NR.sup.8,
C(.dbd.S)S, NR.sup.8(C.dbd.O), NR.sup.8(C.dbd.S)NR.sup.9,
O(C.dbd.O)NR.sup.8, S(.dbd.O).sub.2, Val-Cit-PAB, Val-Ala-PAB,
Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys,
Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB and any combination
thereof.
[0123] Wherein, in this embodiment, R.sup.8 and R.sup.9 are
independently selected from the group consisting of H,
C.sub.1-C.sub.6 alkyls, C.sub.2-C.sub.6 alkenyls, and
C.sub.2-C.sub.6 alkynyls.
[0124] In some embodiments, L is null. When L is null, active agent
Q is connected directly to M.
[0125] In some embodiments, L is independently selected from the
group consisting of:
##STR00037## ##STR00038## ##STR00039##
or is null.
[0126] In some embodiments, L is independently selected from the
groups as follows:
##STR00040## ##STR00041##
or is null.
[0127] A is a targeting moiety. In some embodiments, A is an
antibody; an antibody fragment, surrogate or variant; a protein
ligand; a protein scaffold; a peptide; or a small molecule ligand.
The antibody fragment can be, for example, Fab, Fab', (Fab')2, or
scFv, Fv.
[0128] Examples of the tumor-associated antigens that can be
targeted according to the subject invention include, but are not
limited to, tumor-associated antigens (1) to (36) listed below. The
name, alternative name, and GenBank accession number of each of the
antigen examples is provided. Nucleic acid and protein sequences
corresponding to these exemplary tumor-associated antigens can be
found in public databases such as GenBank.
[0129] (1) BMPR1B (bone morphogenetic protein receptor type-1B,
GenBank accession number NM 001203):
[0130] (2) E16 (LAT1, SLC7A5, GenBank accession number bandit_00:
3486);
[0131] (3) STEAPl (six transmembrane epithelial antigen of
prostate, GenBank accession number NM_012449);
[0132] (4) 0772P (CA125, MUC16, GenBank accession number
AF361486);
[0133] (5) MPF (MPF, MSLN, SMR, megakaryocyte strengthening factor,
mesothelin, GenBank accession number NM_005823);
[0134] (6) Napi3h (NAP1-3B, NPTIlb, SLC34A2, solute carrier family
34 member (sodium phosphate) member 2, type II sodium-dependent
phosphate transporter 3b, GenBank accession number NM_006424);
[0135] (7) Sema5b (FLJ110372, KIAA1445, Mm.42015, SEMA5B, SEMAG,
semaphorin 5b H log, sema domain, seven thrombospondin repeats
(type I and type I), a transmembrane domain (TM) and short
cytoplasmic domain. (semaphorin) 5b, GenBank accession number
AB040878);
[0136] (8) PSCA hlg (2700050C12Rik. C530008016Rik. RIKEN
cDNA2700050C12, RIKENcDNA2700050C12 gene, GenBank accession number
AY358628);
[0137] (9) ETBR (the endothelin B receptor, GenBank accession
number AY275463);
[0138] (10) MSG783 (RNF124, Pseudo protein FLJ20315, GenBank
accession number NM_017763);
[0139] (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP I, STAMP I, STEAP2,
STMP, prostate cancer-related genes 1, prostate cancer-associated
protein 1, six transmembrane epithelial antigen of prostate 2. six
transmembrane prostate protein, GenBank accession number
AF455138);
[0140] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient
receptor potential cation channel, subfamily M, member 4, GenBank
accession number NM_017636);
[0141] (13) CRIPTO (CR, CRI, CRGF, CRIPTO, TDGFI, teratoma-derived
growth factor, GenBank accession number NP_003203 or
NM_003212);
[0142] (14) CD21 (CR2 (complement receptor type 2) or C3DR (C3d/EB
virus receptor) or Hs.73792, GenBank accession number M26004);
[0143] (15) CD79b (CD79B, CD79 .beta., IGb (immunoglobulin related
.beta.), B29, GenBank accession number NM_000626);
[0144] (16) FcRH2 (IFGP4, IRTA4, SPAPIA (comprising phosphatase
anchored proteins Ia of SH2 domains), SPAP1B, SPAP1C, GenBank
accession number NM_030764);
[0145] (17) HER2 (ErbB2, GenBank accession number M11730);
[0146] (18) NCA (CEACAM6, GenBank accession number M18728);
[0147] (19) MDP (DPEPI, GenBank accession number BC017023);
[0148] (20) IL20Ra (IL20Ra, ZCYT0R7, GenBank accession number
AF184971);
[0149] (21) Brevican (BCAN, BEHAB, GenBank accession number
AF229053):
[0150] (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, GenBank accession
number NM_004442);
[0151] (23) ASLG659 (B7h, GenBank accession number AXO92328);
[0152] (24) PSCA (prostate stem cell antigen former body, GenBank
accession number AJ297436);
[0153] (25) GEDA (GenBank accession number AY260763);
[0154] (26) BAFF-R (B cell activating factor receptor, BLyS
receptor 3, BR3, GenBank accession number AF116456);
[0155] (27) CD22 (B-cell receptor CD22-B isoform, GenBank Accession
No. AK026467);
[0156] (28) CD79a (CD79A. CD79a, immunoglobulin-related .alpha.,
capable of covalent interactions with Ig (CD79B) and form a complex
with IgM molecules on the surface, transduced B cells involved in B
cell signal specific protein, GenBank accession number
NP_001774.I);
[0157] (29) CXCR5 (Burkitt's lymphoma receptor 1, a G
protein-coupled receptor chemokine-activated by CXCL13, plays a
role in lymphocyte migration and humoral defense, possibly affects
HIV-2 infection, AIDS, lymphoma, myeloma, and leukemia, GenBank
accession number NP_001701.1);
[0158] (30) HLA-DOB (MHCII molecules of Beta subunit (la antigen)
that binds peptides and presents them to CD4+ T cells in small
lymphoid, GenBank accession number NP_002111.1);
[0159] (31) P2X5 (P2X purinergic receptor ligand-gated ion channel
5, the extracellular ATP-gated ion channels, may be involved in
synaptic transmission and neurogenesis, the defect thereof may
cause pathophysiology of idiopathic detrusor instability, GenBank
accession number NP_002552.2);
[0160] (32) CD72 (B cell differentiation antigen CD72, Lyb-2,
GenBank accession number NP_001773.I);
[0161] (33) LY64 (lymphocyte antigen 64 (RP105), leucine-rich
repeat type I membrane proteins (LRR) family, regulates B cell
activation, apoptosis, and loss of functions, may be involved in
increased activities in patients with systemic lupus erythematosus.
GenBank accession number NP_005573.I);
[0162] (34) FcRHI (Fe 1 receptor-like protein, an immunoglobulin
domain receptor putative Fc, comprises C2 type Ig-like and ITAM
domains, may play a role in B cell differentiation. GenBank
accession No. NP_443170.1);
[0163] (35) IRTA2 (immunoglobulin superfamily translocation
associated receptor 2, putative immune receptor, may play a role in
B cell development and lymphoma development; genetic disorders
caused by a translocation occurs in some B the cell malignancies,
GenBank accession number NP_112571.1);
[0164] (36) TENB2 (putative transmembrane proteoglycan, and
relevant to growth factors EGF/hereguli family and follistatin,
GenBank accession number AF179274).
[0165] In certain embodiments, A is a humanized monoclonal antibody
that targets a tumor-associated antigen. The humanized antibody
may, but is not limited to: Trastuzumab (anti-HER2), Pertuzumab
(anti-HER2), Rituximab (anti-CD20), Alemtuzumab (anti-CD52),
Bevacizumab (anti-VEGF), Adalimumab (anti-TNF-alpha), Cetuximab
(anti-EGFR), Amatuximab (anti-mesothelin), Blinatumomab
(anti-CD19), Brentuximab (anti-CD30), Certolizumab pegol
(anti-TNF-alpha), Panitumumab (anti-EGFR), Nimotuzumab (anti-EGFR),
Gemtuzumab (anti-CD33), Golimumab (anti-TNF-alpha), Ibritumomab
(anti-CD20), Infliximab (anti-TNF-alpha), Ipilimumab (anti-CTLA-4),
Ofatumumab (anti-CD20), and Tositumomab (anti-CD20).
[0166] In some embodiments, the antibody-drug conjugate has the
following structure of Formula II:
##STR00042##
[0167] In some embodiments, the antibody-drug conjugate has the
following structure of Formula II:
##STR00043##
[0168] In some embodiments, the antibody-drug conjugate has the
following structure of Formula II:
##STR00044##
[0169] In some embodiments, the antibody-drug conjugate has the
following structure of Formula II:
##STR00045##
[0170] In some embodiments, the antibody-drug conjugate has the
following structure of Formula II:
##STR00046##
[0171] In some embodiments, the antibody-drug conjugate has the
following structure of Formula II:
##STR00047##
[0172] In some embodiments, the antibody-drug conjugate has the
following structure of Formula II:
##STR00048##
[0173] In some embodiments, the antibody-drug conjugate has the
following structure of Formula II:
##STR00049##
[0174] In some embodiments, the antibody-drug conjugate has the
following structure of Formula II:
##STR00050##
[0175] In some embodiments, the antibody-drug conjugate has the
following structure of Formula II:
##STR00051##
[0176] In some embodiments, the antibody-drug conjugate has the
following structure of Formula II:
##STR00052##
[0177] In some embodiments, the antibody-drug conjugate has the
following structure of Formula II:
##STR00053##
[0178] In certain embodiments, in the above compounds, A includes,
but is not limited to trastuzumab.
Therapeutic Uses
[0179] In certain embodiments, the conjugates of the subject
invention provide treatment for cancers including, but not limited
to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include squamous cell cancer (e.g. epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung and squamous carcinoma
of the lung, cancer of the peritoneum, hepatocellular cancer,
gastric or stomach cancer including gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
oral cancer, liver cancer, bladder cancer, cancer of the urinary
tract, hepatoma, breast cancer including, for example,
HER2-positive breast cancer, colon cancer, rectal cancer,
colorectal cancer, endometrial or uterine carcinoma, salivary gland
carcinoma, kidney or renal cancer, prostate cancer, vulval cancer,
thyroid cancer, hepatic carcinoma, anal carcinoma, penile
carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain
cancer, head and neck cancers, and associated metastases.
[0180] The conjugates of the subject invention can also be used to
deliver active agents for the treatment of a variety of other
conditions including, but not limited to, inflammatory disorders,
autoimmune disorders, disorders of the nervous system, and
cardiovascular disorders.
Diagnostic Uses
[0181] In other embodiments, the conjugate of the subject invention
comprises a detectable entity as the active agent. The detectable
entity may be, for example, a visualization agent. The agent can be
a synthetic or natural product, and capable of reflecting or
emitting light at a wavelength detectable by suitable imaging
and/or diagnostic equipment such as a microscope. Non-limiting
examples of suitable visualization agents include organic dyes,
food dyes, and fluorescence dyes.
[0182] Thus, in one embodiment the subject invention provides a
diagnostic method that involves contacting a sample, suspected of
having an analyte of interest, with the antibody-drug conjugate of
the subject invention, wherein the target moiety selectively binds
to the analyte of interest and Q is a detectable entity and/or is
an entity to which one or more additional entities will bind
wherein said one or more additional entities are, themselves,
detectable entities, and wherein said method further comprises
analyzing the sample to determine the presence of a detectable
entity, which presence would be indicative of the presence of the
analyte of interest. In one embodiment, the analyte is HER2, or
other antigen associated with cancer. The method could be carried
out either in vivo or in vitro.
Biological Activity of the Conjugates
[0183] The conjugates of the subject invention herein can be
evaluated for efficacy and toxicity using known methods. For
example, the toxicology of a particular compound, or of a subset of
the compounds, sharing certain chemical moieties, can be
established by determining in-vitro toxicity towards a cell line,
such as a mammalian, and preferably human, cell line. The results
of such studies are often predictive of toxicity in animals, such
as mammals, or more specifically, humans. Alternatively, the
toxicity of particular compounds in an animal model, such as mice,
rats, rabbits, dogs or monkeys, can be determined using known
methods.
[0184] The efficacy of a particular compound can be established
using art-recognized methods, such as in-vitro methods, animal
models, or human clinical trials. Art-recognized in-vitro models
exist for nearly every class of condition, including the conditions
abated by the compounds disclosed herein, including cancer,
cardiovascular disease, and various immune system dysfunction, and
infectious diseases. Similarly, acceptable animal models can be
used to establish efficacy of chemicals to treat such conditions.
When selecting a model to determine efficacy, a person of ordinary
skill in the art can be guided by the state of the art to choose an
appropriate model, dose, and route of administration, and
regimen.
[0185] The complexes provided herein may be assayed for binding
affinity to, and specificity for, the desired target by any of the
methods conventionally used for the assay of antibodies. The
complexes may be assayed for efficacy as anti-cancer agents by any
of the methods conventionally used for the assay of
cytostatic/cytotoxic agents, such as assays for potency against
cell cultures, xenograft assays, and the like.
[0186] A person of ordinary skill in the art having the benefit of
the current disclosure will have no difficulty, considering that
skill and the literature available, in determining suitable assay
techniques; from the results of those assays, in determining
suitable doses to test in humans as anticancer agents, and, from
the results of those tests, in determining suitable doses to use to
treat cancers in humans.
Formulations and Administration
[0187] The compositions of the subject invention comprise, in
addition to the complexes described herein, a physiologically
acceptable carrier and/or diluent allowing the transport of the
complexes to the target within the animal after administration. The
carrier and/or diluent can generally be any suitable medium by
which the desired purpose is achieved and that does not affect the
conjugates' capability to be directed to the desired target and to
transport the active agent to this target for the desired effect.
Particularly, the carrier and/or diluent should not deteriorate the
pharmacological potency of the active agent and the capability of
the complex to be directed to a desired target within, or on, the
animal body. Preferably, said carrier and/or diluent is/are
selected from the group consisting of water, physiologically
acceptable aqueous solutions containing salts and/or buffers and
any other solution acceptable for administration to an animal. Such
carriers and diluents are well known to a person skilled in this
field and can be, for example, distilled water, de-ionized water,
pure or ultrapure water, saline, phosphate-buffered saline (PBS),
solutions containing usual buffers which are compatible with the
other components of the drug targeting system etc.
[0188] The compositions can be administered to a subject by methods
including, but not limited to, (a) administration through oral
pathways, which administration includes administration in capsule,
tablet, granule, spray, syrup, or other such forms; (b)
administration through non-oral pathways, which administration
includes administration as an aqueous suspension, an oily
preparation or the like or as a drip, suppository, salve, ointment
or the like; administration via injection, subcutaneously,
intraperitoneally, intravenously, intramuscularly, intradermally,
or the like; as well as (c) administration topically, (d)
administration rectally, or (e) administration vaginally, as deemed
appropriate by those of skill in the art for bringing the compound
of the present embodiment into contact with living tissue; and (f)
administration via controlled released formulations, depot
formulations, and infusion pump delivery.
[0189] As further examples of such modes of administration and as
further disclosure of modes of administration, disclosed herein are
various methods for administration of the disclosed compounds and
pharmaceutical compositions including modes of administration
through intraocular, intranasal, and intraauricular pathways.
[0190] In specific embodiments, the complexes of this invention can
be formulated as solutions for intravenous administration, or as
lyophilized concentrates for reconstitution to prepare intravenous
solutions (to be reconstituted, e.g., with normal saline, 5%
dextrose, or similar isotonic solutions). They will typically be
administered by intravenous injection or infusion. A person of
ordinary skill in the art of pharmaceutical formulation, especially
the formulation of anti-cancer antibodies, will have no difficulty,
considering that skill and the literature available, in developing
suitable formulations.
EXAMPLES
[0191] Following are examples that illustrate procedures for
practicing the invention. These examples should not be construed as
limiting.
Example I Synthesis of Linker-Active Agent Conjugates
Example I-1 Synthesis of Compound 9
##STR00054##
[0192] Synthesis of Compound 2
[0193] A solution of 1,3-diamino-2-propanol (3.15 g, 0.035 mol) and
Et.sub.3N (4.85 mL, 0.035 mol) in methanol (120 mL) was heated to
45.degree. C. To the solution, (Boc).sub.2O (17.05 g, 0.078 mol) in
methanol (80 mL) was dropwise added slowly. The reaction solution
was stirred at 45.degree. C. for 30 min. After additional stirring
at room temperature for 1.5 h, the solvent was removed under
reduced pressure. The crude product was extracted with diethyl
ether (200 mL.times.3) and dried over sodium sulfate to yield the
compound 2 (9.94 g, 97.8%) as a white powder. LC-MS m/z (ES.sup.+),
291.19 (M+H).sup.+.
Synthesis of Compound 3
[0194] Tert-butylbromoacetate (5.41 mL, 33.5 mmol) was added to a
solution of Compound 2 (3.89 g, 13.4 mmol) in dry THF (40 mL) at
room temperature. To the solution, sodium hydride (60% dispersion
in mineral oil, 2.42 g, 60.5 mmol) was subsequently added. The
mixture was filtrated after 5 hours. The filtrate was evaporated
and the residue was purified by column chromatography (petroleum
ether:ethyl acetate=10:1 to 5:1) to give the product 3 (3.96 g,
73.1%) as a white solid. LC-MS m/z (ES.sup.+), 405.26
(M+H).sup.+.
Synthesis of Compound 4
[0195] 5 mL of hydrochloric acid was added to a stirred solution of
compound 3 (1.0 g, 2.5 mmol) in 1,4-dioxane (10 mL) at room
temperature. The reaction was completed after 16 hours. Then the
solvent was removed to give a white crude product (384.2 mg,
69.8%). The product 4 could be used in the next step without
purification. LC-MS m/z (ES.sup.+), 221.05 (M+H).sup.+.
Synthesis of Compound 5
[0196] Acryloyl chloride (267 .mu.L, 3.3 mmol) was added to a
solution of compound 4 (242.1 mg, 1.1 mmol) in saturated sodium
bicarbonate solution/THF (v/v=1:1, 20 mL) at 0.degree. C. The
resulting mixture was vigorously stirred at 0.degree. C. After 10
minutes, the solution was allowed to raise to room temperature and
reacted for 3 hours. The mixture was acidified with hydrochloric
acid to adjust pH<4. The mixture was extracted with ethyl
acetate (50 mL.times.2). The organic layers were combined and
washed with saturated sodium chloride solution (40 mL), dried over
anhydrous magnesium sulfate, filtered and removed the solvent. The
solid residue was purified by prep-HPLC to give product 5 as a gray
powder (196.0 mg, 69.6%). LC-MS m/z (ES.sup.+), 257.12
(M+H).sup.+.
Synthesis of Compound 7
[0197] To a stirred solution of compound 5 (25.6 mg, 0.10 mmol) and
DIPEA (16.5 .mu.L, 0.10 mmol) in dry DMF (6 ml) at 0.degree. C. was
added HOBt (14.9 mg, 0.11 mmol) and DIC (13.9 mg, 0.11 mmol). After
15 minutes, compound 6 (20.5 mg, 0.09 mmol) was added to the
solution. The reaction mixture was allowed to warm to room
temperature and stirred overnight. The mixture was diluted with
water (10 mL) and extracted with ethyl acetate (20 mL.times.3). The
combined organic layers were dried over anhydrous magnesium sulfate
and the solvent was removed in vacuo. The crude product was
purified by column chromatography (petroleum ether:ethyl
acetate=3:1) to yield compound 7 as a white solid (24.3 mg, 62.6%).
LC-MS m/z (ES.sup.+), 467.22 (M+H).sup.+.
Synthesis of Compound 9
[0198] Compound 7 (140.1 mg, 0.3 mmol) was dissolved in 5 mL
CH.sub.2Cl.sub.2 and cooled to 0.degree. C. in an ice bath.
Et.sub.3N (1 mg, 0.01 mmol) and the compound 8 (73.7 mg, 0.1 mmol)
were added to the resulting mixture and left to stir for 30
minutes. The solution was allowed to warm to room temperature and
stirred for another 1.5 hours. The solvent was removed under
reduced pressure, and the solid residue was purified by pre-HPLC to
give compound 9 as a white powder (90 mg, 74.8%). LC-MS m/z
(ES.sup.+), 1204.49 (M+H).sup.+.
Example I-2 Synthesis of Compound 17
##STR00055## ##STR00056##
[0199] Synthesis of Compound 11
[0200] Compound 10 (263.3 mg, 1.15 mmol) was dissolved in
CH.sub.2Cl.sub.2 (15 mL), Et.sub.3N (5.9 .mu.L, 0.04 mmol) and the
compound 6 (300.1 mg, 0.39 mmol) was added to the resulting mixture
at 0.degree. C. The solution was allowed to warm to room
temperature and maintained for 5 hours. The solvent was removed in
vacuo and the crude product was purified by prep-HPLC to yield
compound 11 (193.2 mg, 49.7%) as a white solid. LC-MS m/z
(ES.sup.+), 994.43 (M+H).sup.+.
Synthesis of Compound 13
[0201] To a mixture of compound 12 (0.45 g, 4.2 mol) and Et.sub.3N
(8 mL, 0.06 mol) in THF (15 mL), a solution of
2-(tert-Butoxycarbonyloxyimino)-2-phenylacetonitrile (2.1 g, 8.3
mol) in THF (30 mL) was added dropwise at 0.degree. C. Following
complete addition, the solution was allowed to warm to room
temperature and left to stir for 4 hours. The reaction mixture was
concentrated to oil under reduced pressure and CH.sub.2Cl.sub.2 (50
mL) was added. The mixture was washed with sodium hydroxide (5%, 30
mL) and brine (30 mL). The organic layer was dried over sodium
sulfate and concentrated under reduced pressure. The crude product
was purified by column chromatography (silica gel,
MeOH:CH.sub.2Cl.sub.2=1:10, v/v) to give compound 13 as a yellow
oil (803.1 mg, 60.7%). LC-MS m/z (ES.sup.+), 304.22
(M+H).sup.+.
Synthesis of Compound 14
[0202] Succinic anhydride (265.2 mg, 2.65 mmol) was added to a
solution of compound 13 (800.1 mg, 2.65 mmol) in CH.sub.2Cl.sub.2
(10 mL). The mixture was stirred at room temperature overnight and
then concentrated to oil under reduced pressure. The crude product
was purified by column chromatography (silica gel,
MeOH:CH.sub.2Cl.sub.2=1:8, v/v) to give compound 14 (506.5 mg
47.6%) as a gray oil. LC-MS m/z (ES.sup.+), 404.24 (M+H).sup.+.
Synthesis of Compound 15
[0203] Compound 14 (503.5 mg 1.25 mmol) was dissolved in
CH.sub.2Cl.sub.2 (10 mL) along with trifluoroacetic acid (2 mL).
The solution was allowed to react for 3 hours at room temperature.
The solvent was removed under reduced pressure to yield compound 15
as a gray oil (190.2 mg, 75.1%) without further purification. LC-MS
m/z (ES.sup.+), 204.13 (M+H).sup.+.
Synthesis of Compound 16
[0204] To a mixture of potassium carbonate (68.3 mg, 0.5 mmol) in
water (5 mL) and compound 15 (67.2 mg, 0.33 mmol) in ethyl acetate
(10 mL), a solution of acryloyl chloride (53.6 .mu.L, 0.67 mmol) in
ethyl acetate (10 mL) was added dropwise at 0.degree. C. The
resulting mixture was stirred at 0.degree. C. in an ice bath for 10
minutes and then allowed to react at room temperature for 5 hours.
The mixture was acidified with hydrochloric acid to adjust pH<5.
The mixture was extracted with ethyl acetate (20 mL.times.2). The
organic layers were combined and washed with saturated sodium
chloride solution (20 mL), dried over anhydrous magnesium sulfate,
filtered and the solvent removed. The solid residue was purified by
column chromatography (MeOH:CH.sub.2Cl.sub.2=10:1) to give product
16 as a yellow oil (63.1 mg, 61.7%). LC-MS m/z (ES.sup.+), 312.15
(M+H).sup.+.
Synthesis of Compound 17
[0205] Compound 16 (22.2 mg, 0.07 mmol), HOBt (9.7 mg 0.07 mmol)
and DIC (11 .mu.L, 0.07 mmol) were dissolved in 8 mL DMF and cooled
to 0.degree. C. in an ice bath. Subsequently, compound 11 (59.1 mg,
0.06 mmol) and DIPEA (12.4 .mu.L, 0.07 mmol) were added to the
mixture. The solution was allowed to warm to room temperature and
left to react for 24 hours. The resulting mixture was concentrated
and purified by prep-HPLC to yield compound 17 (11.3 mg, 39.5%) as
a white solid. LC-MS m/z (ES.sup.+), 1287.56 (M+H).sup.+.
Example I-3 Synthesis of Compound 25
##STR00057## ##STR00058##
[0206] Synthesis of Compound 20
[0207] To a stirred solution of 18 (191.6 mg, 0.25 mmol) in dry DMF
(15 mL) at 0.degree. C. under nitrogen, HOBt (33.8 mg, 0.25 mmol)
and compound 19 (197.3 mg, 0.26 mmol) was added. After 15 minutes,
pyridine (3 mL) and DIPEA (52.4 .mu.L, 0.30 mmol) were added and
reacted for 30 minutes at 0.degree. C. Then the reaction mixture
was allowed to warm to room temperature and stirred for 3 hours.
The mixture was replenished DIPEA (52.4 .mu.L, 0.30 mmol) and
reacted for 32 hours. Upon completion of the reaction, the solvent
was removed under reduced pressure. The residue was purified by
column chromatography (MeOH:CH.sub.2Cl.sub.2=20:1) to give the
compound 20 (126.4 mg, 37.5%) as a white solid. LC-MS m/z
(ES.sup.+), 1344.77 (M+H).sup.+.
Synthesis of Compound 21
[0208] Diethylamine (4 mL, 38.8 mmol) was added to a solution of
compound 20 (126.4 mg, 0.1 mmol) in DMF (8 mL) at room temperature.
After stirring for 2 hours, the solvent was removed under reduced
pressure to give a crude product 21(104.1 mg, 99%). The product
could be used in the next step without purification. LC-MS m/z
(ES.sup.+), 1123.72 (M+H).sup.+.
Synthesis of Compound 23
[0209] A solution of acryloyl chloride (7.31 mL, 90.00 mmol) in dry
CH.sub.2Cl.sub.2 (50 mL) was added dropwise to a mixture of
compound 22 (2.64 g, 20.00 mmol) and Et.sub.3N (16.60 mL; 120.00
mmol) in dry CH.sub.2Cl.sub.2 (60 mL) at 0.degree. C. under argon.
The mixture was allowed to raise to room temperature and reacted
for 4 hours. Then the mixture was treated with saturated sodium
bicarbonate solution (150 mL) and brine (150 mL), and extracted
with CH.sub.2Cl.sub.2 (150 mL.times.3). The combined organic layers
were dried over anhydrous magnesium sulfate and the solvent was
removed in vacuo. The crude product was purified by column
chromatography (petroleum ether:ethyl acetate=10:1) to afford the
compound 23 as a white solid (4.88 g, 83.0%). LC-MS m/z (ES.sup.+),
295.10 [M+H]+.
Synthesis of Compound 24
[0210] To a solution of compound 23 (444.1 mg, 1.5 mmol) and
Et.sub.3N (0.23 mL, 1.66 mmol) in CH.sub.2Cl.sub.2 (30 mL),
mercapto acetic acid (0.23 mL, 3.32 mmol) in CH.sub.2Cl.sub.2 (5
mL) was added dropwise under an ice bath. Following complete
addition, the solution was allowed to warm to room temperature and
left to stir overnight. The solvent was removed in vacuo and the
residue was purified by column chromatography
(MeOH:CH.sub.2Cl.sub.2=60:1-30:1) to give the compound 24 as a
yellow oil (1.72 g, 36.87%). LC-MS m/z (ES), 385.10
(M-H).sup.-.
Synthesis of Compound 25
[0211] Compound 24 (22.2 mg, 0.057 mmol), HOBt (9.3 mg, 0.069 mmol)
and EDCI (16.5 mg, 0.086 mmol) were dissolved in 5.0 mL
CH.sub.2Cl.sub.2. Subsequently, compound 21 (64.5 mg, 0.063 mmol)
and DIPEA (20.3 .mu.L, 0.104 mmol) were added to the mixture. The
solution was allowed to warm to room temperature and left to react
for 24 hours. The resulting solution was separated by prep-HPLC to
give compound 25 as a white solid (25.3 mg, 29.7%). LC-MS m/z
(ES.sup.+), 1479.80 (M+H).sup.+.
Example I-4 Synthesis of Compound 31
##STR00059## ##STR00060##
[0212] Synthesis of Compound 27
[0213] To a solution of 18 (194.2 mg, 0.25 mmol) in dry DMF (15 mL)
at 0.degree. C. under nitrogen, HOBt (34.2 mg, 0.25 mmol) and
compound 26 (200.3 mg, 0.28 mmol) was added. After stirring for 15
minutes, pyridine (3 mL) and DIPEA (53.1 .mu.L, 0.32 mmol) were
added and left to react for 30 minutes at 0.degree. C. Then the
reaction mixture was allowed to stir at room temperature for 3
hours. The mixture was replenished DIPEA (53.1 .mu.L, 0.32 mmol)
and reacted for another 24 hours. Upon completion of the reaction,
the mixture was concentrated in vacuo. The crude product was
purified by column chromatography to give the compound 27 (126.2
mg, 37.0%) as a white solid. LC-MS m/z (ES.sup.+), 1398.75
(M+H).sup.+.
Synthesis of Compound 28
[0214] Diethylamine (4 mL) was added to a solution of compound 27
(126.2 mg, 0.09 mmol) in DMF (8 mL) at room temperature. After
stirring for 2.5 hours, the solvent was removed under reduced
pressure to give a crude product 28 (73.3 mg, 68.9%) which could be
used without purification. LC-MS m/z (ES.sup.+), 1176.68
(M+H).sup.+.
Synthesis of Compound 30
[0215] To a solution of compound 29 (1.87 g, 7.5 mmol) and
Et.sub.3N (104 .mu.L, 0.75 mmol) in CH.sub.2Cl.sub.2 (40 mL),
mercapto acetic acid (103.9 .mu.L, 1.5 mmol) in CH.sub.2Cl.sub.2
(10 mL) was dropwise added at 0.degree. C. Upon complete addition,
the solution was allowed to warm to room temperature and left to
stir overnight. The solvent was removed in vacuo and the residue
was purified by column chromatography to give the compound 30 as a
white solid (1.72 g, 36.87%). LC-MS m/z (ES), 342.11
(M-H).sup.-.
Synthesis of Compound 31
[0216] Compound 30 (22.2 mg 0.065 mmol), HOBt (8.8 mg 0.065 momol)
and DIC (11 .mu.L, 0.065 mmol) were added to DMF (8 mL) and cooled
to 0.degree. C. in an ice bath. Compound 28 (63.7 mg, 0.054 mmol)
and DIPEA (12.4 .mu.L, 0.054 mmol)) were added to the solution. The
mixture was allowed to warm to room temperature and left to react
for 24 hours. The residue was purified by column chromatography
(MeOH:CH.sub.2Cl.sub.2=60:1-30:1) to yield compound 31 as a white
solid (11.3 mg, 39.5%). LC-MS m/z (ES.sup.+), 1499.78
(M+H).sup.+.
Example I-5 Synthesis of Compound 36
##STR00061##
[0217] Synthesis of Compound 33
[0218] 2,4-Diaminobenzoic acid 32 (501.8 mg, 3.3 mmol) was
dispersed in ethyl acetate (20 mL) and a solution of potassium
carbonate (9.0 g, 66 mmol) in water (20 mL) was added. To the
mixture, acryloyl chloride (1 mL, 13 mmol) was carefully added,
resulting in the formation of a light brown precipitate. The
reacting mixture was then allowed to reach room temperature, lead
to the dissolution of the precipitate. After 2 hour, the reaction
was complete. The organic layer was discarded and the aqueous layer
was acidified with 5% hydrochloric acid, making a precipitate
appear at pH<4. The precipitate was filtered, washed with
petroleum ether and diluted with ethyl acetate. The organic layer
was then dried over anhydrous magnesium sulfate, filtered, and
removed in vacuo. The solid residue was suspended in water,
filtered, and washed thoroughly with diethyl ether to give product
33 (670.2 mg, 78%) as a gray powder. LC-MS m/z (ES.sup.+), 261.09
(M+H).sup.+.
Synthesis of Compound 34
[0219] Compound 33 (260.1 mg, 1.0 mmol) and EDCI (210.9 mg, 1.1
mmol), N-hydroxy succinimide (126.6 mg, 1.1 mmol) was dissolved in
DMF (14 mL). The solution was stirred overnight at room
temperature. Upon completion of the reaction, the solvent was
removed under reduced pressure. The solid residue was purified by
column chromatography with CH.sub.2Cl.sub.2/MeOH (10:1) as eluent
to give the product 34 as a light brown solid (273 mg, 76.4%).
LC-MS m/z (ES.sup.+), 358.11 (M+H).sup.+.
Synthesis of Compound 35
[0220] To a solution of 4-aminobutyric acid (82.4 mg, 0.8 mmol) and
DIPEA (489 .mu.L, 2.8 mmol) in DMF (10 mL), a solution of compound
34 (260.7 mg, 0.73 mmol) in DMF (10 mL) was added at 0.degree. C.
in an ice bath. The mixture was stirred for 10 minutes at 0.degree.
C. and then stirred at room temperature overnight. The solvent was
removed in vacuo when the reaction was completed. The solid residue
was purified by column chromatography (CH.sub.2Cl.sub.2: MeOH=10:1,
v/v) to yield the compound 35 (192.3 mg, 76.2%). LC-MS m/z
(ES.sup.+), 346.14 (M+H).sup.+.
Synthesis of Compound 36
[0221] HOBt (14.9 mg, 0.11 mmol), EDCI (21.1 mg, 0.11 mmol) and
DIPEA (19.2 .mu.L, 0.11 mmol) was added to a stirred solution of 35
(34.5 mg, 0.1 mmol) in dry DMF (10 ml) at 0.degree. C. After 15
minutes, compound 21 (112.3 mg, 0.1 mmol) was added. The reaction
mixture was allowed to warm to room temperature and stirred for 24
hours. The mixture was added water (15 mL) and extracted with ethyl
acetate (20 mL.times.3). The combined organic layers were dried
over anhydrous magnesium sulfate, filtered, and removed in vacuo.
The crude product was purified by column chromatography
(CH.sub.2Cl.sub.2: MeOH=10:1, v/v) to afford compound 36 as a white
solid (56.5 mg, 39%). LC-MS m/z (ES.sup.+), 1450.84
(M+H).sup.+.
Example I-6 Synthesis of Compound 43
##STR00062##
[0222] Synthesis of Compound 38
[0223] Into a 100 mL round-bottom flask was placed a solution of
compound 37 (1.05 g, 6.9 mmol) and DMAP (1.94 g, 15.9 mmol) in
CH.sub.2Cl.sub.2 (20 mL), acryloyl chloride (1.38 g, 15.2 mmol) was
added dropwise with stirring at 0.degree. C. The reaction was
stirred for 1 hour at room temperature. The reaction was quenched
by the addition of water (50 mL) and extracted with
CH.sub.2Cl.sub.2 (20 mL.times.3). The organic phase was combined,
washed with saturated sodium bicarbonate solution (30 mL), and
brine (30 mL), dried over anhydrous sodium sulfate, and filtered.
The filtrate was concentrated under reduced pressure to afford
compound 38 (1.51 g, 85.8%) as a light yellow semi-solid. LC-MS m/z
(ES.sup.+), 253.04 (M+H).sup.+.
Synthesis of Compound 39
[0224] Compound 38 (1.56 g, 5.94 mmol) and 4-aminobutanoic acid
(673.0 mg, 6.53 mmol) were dissolved in EtOH (15 ml) along with
sodium hydroxide (713.3 mg, 17.82 mmol) in water (15 mL). The
reaction mixture was stirred for 2 hours at 80.degree. C. After
cooled to room temperature, the solvent was concentrated under
reduced pressure. The mixture was acidify to ph<7 with
hydrochloric acid (0.5 N). The solid was collected by filtration,
washed with water (10 mL.times.2) and dried to afford compound 39
(490.2 mg, 25.8%) as an off-white solid. LC-MS m/z (ES.sup.+),
320.03 (M+H).sup.+.
Synthesis of Compound 41
[0225] Compound 39 (230.1 mg, 0.72 mmol), HOBt (116.7 mg, 0.86
mmol), EDCI (151.8 mg, 0.79 mmol) and DIPEA (279.2 mg, 2.2 mmol)
were added to DMF (20 mL) and stirred at 0.degree. C. in an ice
bath. After 1 hour, compound 40 (149.1 mg, 0.79 mmol) was added to
the solution. The mixture was allowed to warm to room temperature
and left to react for 18 hours. The reaction mixture was diluted
with brine (50 mL), extracted with ethyl acetate (20 mL.times.3).
The organic layers were combined and dried with anhydrous sulfate
sodium, filtered and concentrated. The residue was purified by
column chromatography (MeOH:CH.sub.2Cl.sub.2=200/1.about.10/1, v/v)
to yield compound 41 as a yellow solid (97.3 mg, 27.6%). LC-MS m/z
(ES.sup.+), 490.23 (M+H).sup.+.
Synthesis of Compound 43
[0226] A solution of compound 41 (90.1 mg, 0.18 mmol), EEDQ (54.6
mg, 0.22 mmol) in anhydrous CH.sub.2Cl.sub.2 (7 ml) and anhydrous
MeOH (0.07 mL) was stirred for 1 hour at room temperature.
Subsequently, compound 42 (133.5 mg, 0.184 mmol) was added. The
reaction mixture was stirred for additional 18 hours. The reaction
mixture was concentrated in vacuo, and purified by prep-HPLC to
afford compound 43 (12.3 mg, 5.6%) as a white solid. LC-MS m/z
(ES.sup.+), 1197.51 (M+H).sup.+.
Example I-7 Synthesis of Compound 49
##STR00063##
[0227] Synthesis of Compound 45
[0228] To a solution of compound 44 (1.13 g, 6.53 mmol) in methanol
(30 mL), hydrochloric acid (gas) was introduced in with stirring at
0.degree. C. The reaction was stirred for 2 hours at 0.degree. C.
The mixture was concentrated and the residue was dissolved in water
(50 mL). The mixture was adjust to 8 with saturated sodium
bicarbonate, extracted with ethyl acetate (20 mL.times.3). The
organic layer was combined and dried with anhydrous sodium sulfate,
filtered. The filtrate was concentrated to afford compound 45
(710.1 mg, 65.1%) as a yellow solid. LC-MS m/z (ES.sup.+), 168.07
(M+H).sup.+.
Synthesis of Compound 46
[0229] Acryloyl chloride (947.1 mg, 10.47 mmol) was added dropwise
to a solution of compound 45 (700.5 mg, 4.19 mmol) in pyridine (20
mL) with stirring at 0.degree. C. in an ice bath. The reaction
mixture was stirred for 2 hours at 100.degree. C. After cooled to
room temperature, the reaction mixture was concentrated under
reduced pressure. The residue was purified by column chromatography
(MeOH:CH.sub.2Cl.sub.2=200/1.about.10/1, v/v) to afford compound 46
(520.4 mg, 45.2%) as a yellow solid. LC-MS m/z (ES.sup.+), 276.09
(M+H).sup.+.
Synthesis of Compound 47
[0230] A mixture of compound 46 (510.2 mg, 1.85 mmol), sodium
hydroxide (370.6 mg, 9.26 mmol), THF (10 mL) and water (10 mL) was
stirred for 4 hours at room temperature. Upon complete reaction,
the mixture was concentrated and adjusted PH to 7 with hydrochloric
acid (IN). The compound 47 (360.2 mg, 74.1%) was collected by
filtration as an off-white solid. LC-MS m/z (ES.sup.+), 262.07
(M+H).sup.+.
Synthesis of Compound 48
[0231] Compound 47 (120.5 mg, 0.5 mmol), HOBt (4.7 mg, 0.6 mmol),
EDCI (96.8 mg, 0.5 mmol) and DIPEA (178.1 mg, 1.4 mmol) were added
to DMF (10 mL) and stirred at 0.degree. C. in an ice bath After 1
hour, compound 40 (95.1 mg, 0.5 mmol) was added to the solution.
The mixture was allowed to warm to room temperature and left to
react for 18 hours. The reaction mixture was diluted with brine (20
mL), extracted with ethyl acetate (10 mL.times.3). The organic
layers were combined and dried with anhydrous sulfate sodium,
filtered and concentrated. The residue was purified by column
chromatography (MeOH:CH.sub.2Cl.sub.2=100/1.about.20/1, v/v) to
yield compound 48 as an off white solid (71 mg, 35.8%). LC-MS m/z
(ES.sup.+), 432.07 (M+H).sup.+.
Synthesis of Compound 49
[0232] Compound 48 (60.2 mg, 0.14 mmol), EEDQ (41.3 mg, 0.17 mmol)
was dissolved in anhydrous CH.sub.2Cl.sub.2 (6 ml) and anhydrous
MeOH (0.06 mL) and left to stir for 1 hour at room temperature.
Compound 42 (100.9 mg, 0.14 mmol) was added to the solution. The
reaction mixture was stirred for additional 18 hours. The reaction
mixture was concentrated under reduced pressure, and purified by
prep-HPLC to afford compound 49 (12.4 mg, 7.61%) as a white solid.
LC-MS m/z (ES.sup.+), 1139.45 (M+H).sup.+.
Example I-8 Synthesis of Compound 55
##STR00064## ##STR00065##
[0233] Synthesis of Compound 51
[0234] Et.sub.3N (332.7 .mu.L, 2.4 mmol) was added to a solution of
compound 4 (440.0 mg, 2 mmol) in MeOH (10 mL) at 45.degree. C. Then
a solution of (Boc).sub.2O (436.2 mg, 2 mmol) in MeOH (10 mL) was
added. The resulting mixture was vigorously stirred at 45.degree.
C. for 30 minutes, followed by stirring at room temperature for 2
hours. After solvent removed, the residue was washed with
hydrochloric acid (5%, 10 mL), saturated sodium chloride solution
(15 mL), and extracted with ethyl acetate (10 mL.times.3). The
organic layer was dried over anhydrous magnesium sulfate, filtered,
and removed in vacuo. The solid residue was recrystallized by
petroleum ether/ethyl acetate (10 mL, v:v=2:1,) to give compound 50
(375.4 mg, 75.6%) as a white powder. LC-MS m/z (ES.sup.+), 249.15
(M+H).sup.+.
Synthesis of Compound 51
[0235] Acryloyl chloride (121 .mu.L, 1.5 mmol) was added to a
solution of compound 50 (248.0 mg, 1.0 mmol) in saturated sodium
bicarbonate solution/THF (v/v=1:1, 20 mL) at 0.degree. C. The
resulting mixture was vigorously stirred at 0.degree. C. for 10
minutes. The mixture was allowed to raise to room temperature and
reacted for 3 hours. The mixture was acidified with hydrochloric
acid to adjust pH<4. The mixture was extracted by ethyl acetate
(20 mL.times.3). The organic layers were combined and washed with
saturated sodium chloride solution (30 mL), dried over anhydrous
magnesium sulfate, filtered and the solvent was removed. The solid
residue was purified by prep-HPLC to give product 51 (176.1 mg,
58.3%) as a gray oil. LC-MS m/z (ES.sup.+), 303.16 (M+H).sup.+.
Synthesis of Compound 52
[0236] Compound 51 (30.2 mg, 0.1 mmol), HOBt (14.7 mg, 0.11 mmol)
and DIC (13.9 mg, 0.11 mmol) were dissolved in dry DMF (6 mL) and
cooled to 0.degree. C. in an ice bath. After 15 minutes, compound 6
(20.5 mg, 0.09 mmol) and DIPEA (19.2 .mu.L, 0.11 mmol) were added
to the mixture. The solution was allowed to warm to room
temperature and stirred overnight. The mixture was diluted with
water (10 mL) and extracted with ethyl acetate (20 mL.times.3). The
combined organic layers were dried over anhydrous magnesium sulfate
and the solvent was removed in vacuo. The crude product was
purified by column chromatography (CH.sub.2Cl.sub.2: MeOH=20:1,
v/v) to yield compound 52 as a white solid (28.2 mg, 60.7%). LC-MS
m/z (ES.sup.+), 513.26 (M+H).sup.+.
Synthesis of Compound 53
[0237] A solution of Compound 52 (28.2 mg, 0.06 mmol)
trifluoroacetic acid (1.0 mL) in CH.sub.2Cl.sub.2 (5 mL) was
stirred for 3 hours at room temperature. The solvent was removed
under reduced pressure to yield compound 53 (20.4 mg, 88.2%) as a
gray solid which could be used in next step without further
purification. LC-MS m/z (ES.sup.+), 413.21 (M+H).sup.+.
Synthesis of Compound 54
[0238] Compound 53 (41.2 mg, 0.1 mmol) was dissolved in 10 mL DMF
and cooled to 0.degree. C. in an ice bath. Et.sub.3N (6.9 .mu.L,
0.05 mmol) and the compound 8 (73.7 mg, 0.1 mmol) were added
dropwise to the solution and left to stir for 30 minutes. The
solution was allowed to warm to room temperature and stirred for
another 5 hours. The mixture was concentrated under reduced
pressure, and the residue was purified by pre-HPLC to give compound
54 (73 mg, 63.6%) as a white powder. LC-MS m/z (ES.sup.+), 1148.47
(M+H).sup.+.
Synthesis of Compound 55
[0239] Bromoacetyl bromide (150.0 mg, 0.75 mmol) was added to a
solution of compound 54 (573.7 mg, 0.5 mmol) in DMF (10 mL) at
0.degree. C. The resulting mixture was stirred at 0.degree. C. for
10 minutes and then allowed to stir at room temperature for another
3 hours. The mixture was added water (20 mL) and extracted by ethyl
acetate (20 mL.times.3). The combined organic layers were
concentrated, and the residue was purified by pre-HPLC to give
compound 55 (421 mg, 66.4%) as a yellow powder. LC-MS m/z
(ES.sup.+), 1270.38 (M+H).sup.+.
Example I-9 Synthesis of Compound 59
##STR00066##
[0240] Synthesis of Compound 56
[0241] To a mixture of potassium carbonate (51.2 mg, 0.37 mmol) in
water (5 mL) and compound 15 (50.0 mg, 0.25 mmol) in ethyl acetate
(10 mL), a solution of acryloyl chloride (16 .mu.L, 0.2 mmol) in
ethyl acetate (8 mL) was dropwise added slowly at 0.degree. C. Upon
complete addition, the resulting mixture was stirred at 0.degree.
C. in an ice bath for additional 10 minutes. Then the mixture was
allowed to raise to room temperature and reacted for 5 hours. The
mixture was acidified with hydrochloric acid to adjust pH<5. The
mixture was extracted with ethyl acetate (15 mL.times.3). The
organic combined layers were washed with saturated sodium chloride
solution (15 mL), dried over anhydrous magnesium sulfate, filtered
and removed the solvent. The solid residue was purified by column
chromatography (MeOH: CH.sub.2Cl.sub.2=8:1) to give product 56
(26.2 mg, 40.1%) as a gray oli. LC-MS m/z (ES.sup.+), 258.14
(M+H).sup.+.
Synthesis of Compound 57
[0242] Compound 56 (26.0 mg, 0.10 mmol) was stirred in CHCl.sub.3
(10 mL), maleic anhydride (28.1 mg, 0.11 mmol) was added to the
mixture. The reaction was heated to reflux for 12 hours. After
cooled to room temperature, solid product precipitated was filtered
and washed with CHCl.sub.3 (30 mL.times.3) to afford the compound
57 (20.1 mg, 59.1%) as a white solid. LC-MS m/z (ES.sup.+), 356.14
(M+H).sup.+.
Synthesis of Compound 58
[0243] A solution compound 57 (20.1 mg 0.054 mmol) and sodium
acetate (6.3 mg, 0.047 mmol) in acetic anhydride (10 mL) was heated
to 100.degree. C. for 2 hours. Once the reaction was complete, the
reaction solution was slowly poured into crushed ice with stirring,
and iced water was added. Solid was precipitated out after 1 hour
stirring. The solid residue was filtered and washed with iced water
(15 mL.times.3) to give the compound 58 (9.1 mg, 46.1%) as a white
solid. LC-MS m/z (ES), 338.13 (M+H).sup.+.
Synthesis of Compound 59
[0244] Compound 58 (8.1 mg 0.02 mmol), HOBt (3.25 mg, 0.02 mmol)
and DIC (3.7 .mu.L, 0.02 mmol) were dissolved in 5 mL DMF and
cooled to 0.degree. C. in an ice bath. Subsequently, compound 11
(20.1 mg, 0.02 mmol) and DIPEA (4.1 .mu.L, 0.02 mmol) were added to
the mixture. The solution was allowed to warm to room temperature
and left to react for 24 hours. The resulting mixture was
concentrated and purified by prep-HPLC to yield compound 59 (9.3
mg, 34.9%) as a white solid. LC-MS m/z (ES.sup.+), 1313.53
(M+H).sup.+.
Example I-10 Synthesis of Compound 64
##STR00067##
[0245] Synthesis of Compound 60
[0246] To a solution of compound 32 (140.0 mg, 0.9 mmol) in DMF (6
mL), maleic anhydride (89.8 mg, 0.9 mmol) was added dropwise. The
mixture was stirred at 60.degree. C. for 6 hours and then
concentrated to oil under reduced pressure. The compound 60 (white
solid, 209.1 mg, 90.7%) was obtained by recrystallization from
ethyl acetate (5 ml). LC-MS m/z (ES.sup.+), (M+H).sup.+251.06.
Synthesis of Compound 61
[0247] To a mixture of potassium carbonate (201.3 mg, 1.4 mmol) in
water (15 mL) and compound 60 (209.1 mg, 0.9 mmol) in ethyl acetate
(20 mL), a solution of acryloyl chloride (90 .mu.L, 1.1 mmol) in
ethyl acetate (10 mL) was added dropwise at 0.degree. C. Upon
complete addition, the mixture was allowed to warm to room
temperature and reacted for 5 hours. The mixture was acidified with
hydrochloric acid to adjust pH<5. The mixture was extracted with
ethyl acetate (20 mL.times.2). The organic layers were combined and
washed with saturated sodium chloride solution (20 mL), dried over
anhydrous magnesium sulfate, and filtered. The solvent was removed
under in vacuo to give compound 61 (201.0 mg, 82.7%) as a gray
solid which could be used in next step without further
purification. LC-MS m/z (ES.sup.+), (M+H).sup.+304.07.
Synthesis of Compound 62
[0248] To a solution of crude compound 61 (201.0 mg, 0.7 mmol) in
acetic anhydride (30 mL), sodium acetate (60.5 mg, 0.7 mmol) was
added. The mixture was stirred at 90.degree. C. for 3 hours. The
reaction mixture was poured into ice water and stirred for 30 min
and extracted with ethyl acetate (20 mL.times.3). The combined
organic layers were concentrated and purified by prep-HPLC to give
compound 62 (92.2 mg, 48.6%) as a white solid. LC-MS m/z
(ES.sup.+), (M+H).sup.+287.06.
Synthesis of Compound 64
[0249] The compound 62 (57.0 mg, 0.2 mmol), HOBt (27.2 mg, 0.2
mmol), DIC (29.3 .mu.L, 0.2 mmol) and DIPEA (0.2 mmol, 33.1 .mu.L)
were added to DMF (6 mL). After 30 min, the compound 63 (109.8 mg,
0.15 mmol) and was added at 0.degree. C. The mixture was allowed to
stir at room temperature for 24 hours. The solution was
concentrated and purified by prep-HPLC to give compound 64 (94.8
mg, 47.6%) as a white solid. LC-MS m/z (ES.sup.+), 1000.53
(M+H).sup.+.
Example I-11 Synthesis of Compound 69
##STR00068##
[0250] Synthesis of Compound 66
[0251] To a mixture of compound 65 (145.0 mg, 1.0 mmol) in DMF (6
mL), maleic anhydride (89.8 mg, 0.9 mmol) was dropwise added
slowly. The mixture was heated to reflux for 12 hours. After cooled
to room temperature, solid product precipitated was filtered and
washed with CH.sub.2Cl.sub.2 (20 mL.times.3) to afford the compound
66 (110.2 mg, 45.1%) as a white solid. LC-MS m/z (ES.sup.+), 244.02
(M+H).sup.+.
Synthesis of Compound 67
[0252] A mixture of compound 66 (110.2 mg, 0.5 mmol) and sodium
acetate (14.2 mg, 0.2 mmol) in acetic anhydride (10 mL) was heated
to reflux for 6 hours. Once the reaction was complete, the reaction
mixture was slowly poured into crushed ice with stirring, and iced
water was added. Solid residue was precipitated out after 1 hour
stirring. The solid residue was filtered and washed with iced water
(10 mL.times.3) to give the compound 67 (87.3 mg, 85.7%) as a white
solid. LC-MS m/z (ES.sup.+), 226.01 (M+H).sup.+.
Synthesis of Compound 68
[0253] Succinic anhydride (38.8 mg, 0.4 mmol) was added to a
mixture of compound 67 (87.3 mg, 0.4 mmol) in DMF (6 mL). The
mixture was stirred at 80.degree. C. overnight and then
concentrated to oil under reduced pressure. The crude product was
purified by prep-HPLC to give compound 68 (45.0 mg, 35.7%) as white
solid. LC-MS m/z (ES.sup.+), 326.02 (M+H).sup.+.
Synthesis of Compound 69
[0254] Compound 68 (45.0 mg, 0.14 mmol), HOBt (19.0 mg, 0.14 mmol),
DIC (20.5 .mu.L, 0.14 mmol) and DIPEA (23.2 .mu.L, 0.14 mmol,) were
added to DMF (10 mL) and stirred at 0.degree. C. in an ice bath for
1 hour. Compound 63 (76.9 mg, 0.11 mmol) was added to the solution.
The mixture was allowed to warm to room temperature and left to
react for 36 hours. The reaction mixture was purified by prep-HPLC
to yield compound 69 (64.3 mg, 44.3%) as a white solid. LC-MS m/z
(ES.sup.+), 1039.49 (M+H).sup.+.
Example I-12 Synthesis of Compound 74
##STR00069##
[0255] Synthesis of Compound 71
[0256] Compound 69 (1.0 g, 6.10 mmol) and 4-aminobutanoic acid
(691.9 mg, 6.71 mmol) and triethylamine (3.09 g, 30.5 mmol) was
dissolved in n-Butanol (30 mL). The reaction mixture was stirred
for 18 hours at 90.degree. C. After cooled to room temperature, the
solvent was concentrated under reduced pressure. The residue was
diluted with water (100 mL) and extracted with ethyl acetate (30
mL.times.3). The organic phase was combined and dried over
anhydrous sulfate sodium, filtered and concentrated. The residue
was purified by column chromatography
(MeOH:CH.sub.2Cl.sub.2=200/1.about.10/1, v/v) to yield compound 71
as a yellow solid (482.3 mg, 34.3%). LC-MS m/z (ES.sup.+), 231.06
(M+H).sup.+.
Synthesis of Compound 72
[0257] Acryloyl chloride (947.2 mg, 10.47 mmol) was added dropwise
to a solution of compound 71 (450.5 mg, 1.95 mmol) and TEA (590.8
mg, 5.85 mmol) in CH.sub.2Cl.sub.2 (20 ml) with stirring at
0.degree. C. in an ice bath. The reaction mixture was stirred for
18 hours at room temperature. The reaction was quenched by the
addition of water (50 mL) and extracted with CH.sub.2Cl.sub.2 (20
mL.times.3). The organic layer was combined, washed with brine (30
mL), dried over anhydrous sulfate sodium, filtered and concentrated
to afford compound 72 (255.4 mg, 46%) as a yellow solid. LC-MS m/z
(ES.sup.+), 285.07 (M+H).sup.+.
Synthesis of Compound 73
[0258] Compound 3 (155.7 mg, 0.547 mmol), HOBt (88.7 mg, 0.656
mmol), EDCI (115.4 mg, 0.602 mmol) and DIPEA (212.1 mg, 1.64 mmol)
were added to DMF (10 mL) and stirred at 0.degree. C. in an ice
bath for 1 hour. Compound 40 (113.3 mg, 0.602 mmol) was added to
the solution. The mixture was allowed to warm to room temperature
and left to react for 18 hours. The reaction mixture was diluted
with brine (30 mL), extracted with CH.sub.2Cl.sub.2 (10
mL.times.3). The organic layer was combined and dried over
anhydrous sulfate sodium, filtered and concentrated. The residue
was purified by column chromatography
(MeOH:CH.sub.2Cl.sub.2=200/1.about.10/1, v/v) to yield compound 73
as a yellow solid (90.2 mg, 36.2%). LC-MS m/z (ES.sup.+), 455.17
(M+H).sup.+.
Synthesis of Compound 74
[0259] A solution of compound 73 (85.4 mg, 0.19 mmol), EEDQ (55.4
mg, 0.23 mmol) in anhydrous CH.sub.2Cl.sub.2 (7 ml) and anhydrous
MeOH (0.07 mL) was stirred for 1 hour at room temperature.
Subsequently, compound 42 (135.7 mg, 0.19 mmol) was added. The
reaction mixture was stirred for additional 18 hours. The reaction
mixture was concentrated in vacuo, and purified by prep-HPLC to
afford compound 74 (19.1 mg, 6.7%) as a white solid. LC-MS m/z
(ES.sup.+), 1162.45 (M+H).sup.+.
TABLE-US-00001 TABLE 1 Other linker-active agent conjugates MS ID
Structure of linker-active agent conjugates (M + H).sup.+ 75
##STR00070## 1446.80 76 ##STR00071## 1041.62 77 ##STR00072##
1234.58 78 ##STR00073## 1444.89 79 ##STR00074## 1419.85 80
##STR00075## 1237.25
Example II Preparation of Antibody Drug Conjugates
General Conjugation Procedure
[0260] DTPA (1 mM) and 3.0-5.0 equiv. of TCEP (10 mM) were added to
antibody (10 mg/mL) in a PCR tube, and mixer for 1 hour to reduce
the disulfide bonds on the antibody. Various amounts of the
linker-active agent conjugate were added to the reaction mixtures
and shaken overnight using a vortex mixer. All reactions were
carried out at room temperature. The resultant products were
analyzed by gel electrophoretic (SDS-PAGE) and HIC (Hydrophobic
Interaction Chromatography)-HPLC.
[0261] The antibody drug conjugate was analyzed by reduced
SDS-PAGE: samples mixed with buffer were loaded onto 8%-15%
acrylamide gels, and gel photographs were taken with a UVP
BioDoc-it imaging system.
[0262] The bands (.gtoreq.50 kDa) appeared in lanes 2 and 3, which
suggest that thiol-bridge conjugation was effective (H, L). Bands
at 75 kDa represent the part composed of a heavy and a light chain
which were linked by one pair of bridged thiols (LH). Two heavy
chains were linked by one or two pairs of bridged thiols, which
formed the bands at 100 kDa (HH). Bands at 125 kDa represent the
part in which only one bridge between a heavy and a light chain was
not formed (LHH). Lastly, bands at 150 kDa revealed formation of
fully bridged antibody (LHHL).
[0263] The antibody drug conjugate was analyzed by HIC-HPLC under
conditions: HPLC Column: TSK-GEL Butyl-NPR column 4.6*35 mm; Buffer
A: 75% sodium phosphate (20 mM, pH 7.0), 25% isopropanol (v/v);
Buffer B: sodium phosphate (20 mM, pH 7.0), Ammonium sulfate (1.5
mM); flow rate: 1.0 mL/min.
In Vitro Evaluation of Antibody Drug Conjugates
[0264] On the first day, a tumor cell line of human ovarian cancer
cell lines, such as OVCAR3, was plated at 5000 cells in 100 .mu.l
culture medium on a 96 well culture plate (Cellstar). The cells
were incubated in a CO2 incubator with saturated water at
37.degree. C. overnight. On the second day, two covalent thiol
conjugated ADCs of MSL-31 and MSL-75, were serially diluted
conjugates were added to the 96 well plate containing OVCAR3 cells,
100 .mu.l per well. The initial conjugate was 100000 ng/ml and
diluted to 0.001 ng/ml. OVCAR3 cells with added ADC were incubated
at 37.degree. C. for 72 hours. The viability of tumor cell treated
covalent thiol conjugated ADC was then measured using a cell
viability kit, Cell Counting Kit-8 (DOJINDO, CK04) according to the
manufacturer protocol on a plate reader (BIO-RAD iMark SpectraMax L
from Molecular device). The IC50 value, 50% inhibition of cell
growth was calculated using a curve fitting software, Graphpad.
Example II-1 Preparation of MSL-C31
[0265] DTPA (1 mM) and 5.0 equiv. of TCEP (10 mM) were added to
anti-MSL (10 mg/mL) in a PCR tube. The mixture was agitated with a
vortex mixer for 1 hour to reduce the disulfide bonds on the
antibody. 25% of DMSO was subsequently added to the reaction
mixtures. Compound 31 (5.0 equiv., 10 mM) was added to the reaction
mixtures and shaken overnight using a vortex mixer. All reactions
were carried out at room temperature. After reaction was over, the
mixture was loaded into the Millipore 4 mL centrifugal
ultrafiltration tube, and washed three times with PBS, then
collected the reaction mixtures. FIG. 2 shows that products (the
right lane) of covalent conjugation of at least one pair of
sulfhydryl group will migrate slower than those of heavy (H) or
light chain (L) alone (shown in left lane). The covalent
conjugation between the heavy and light chains produced the band of
75 kDa (HL); the covalent conjugation of two heavy chains produced
the band of 100 kDa (HH). There are 3 peaks in the HIC-HPLC
chromatogram, representing 3, 4, and 5 of DAR, respectively. The
DAR of the resultant MSL-C31 was 3.60 based on the results of
HIC-HPLC (FIG. 3). FIG. 2 shows the result of reduced SDS-PAGE for,
a) naked antibody, b) MSL-C31, the product of the covalent
thiol-conjugate.
[0266] FIG. 3 shows an HIC-HPLC chromatogram of MSL-C31
Example II-2 Preparation of MSL-C75
[0267] DTPA (1 mM) and 5.0 equiv. of TCEP (10 mM) were added to
anti-MSL (10 mg/mL) in a PCR tube. The mixture was agitated with a
vortex mixer for 1 hour to reduce the disulfide bonds on the
antibody. 25% of DMSO was subsequently added to the reaction
mixtures. 5.0 equiv. of Compound 75 (5.0 equiv., 10 mM) was added
to the reaction mixtures and shaken overnight using a vortex mixer.
All reactions were carried out at room temperature. After reaction
was over, the mixture was loaded into the Millipore 4 mL
centrifugal ultrafiltration tube, and washed three times with PBS,
then collected the reaction mixtures. The samples were subjected to
analysis by electrophoresis. The results of reduced SDS-PAGE were
presented at Figure-4. The left lane was naked antibody and right
lane was covalent conjugate of MSL-C75. The DAR of the resultant
MSL-C75 was 3.87 based on HIC-HPLC.
[0268] FIG. 4 shows the results of reduced SDS-PAGE of MSL-C75
[0269] FIG. 5 shows a HIC-HPLC chromatogram of MSL-C75
Table-2 shows IC50 values of two samples: ADC-C31 and ADC-C75,
tested in human ovarian cancer cells.
TABLE-US-00002 TABLE 2 IC50 values of two samples Samples Cell Type
IC50 (ng/mL) MSL-C31 OVCAR3(MSL+) 37.38 MSL-C75 OVCAR3(MSL+)
50.00
Example II-3 Preparation of CD59-C78
[0270] DTPA (1 mM) and 5.0 equiv. of TCEP (10 mM) were added to
anti-CD59 (10 mg/mL) in a PCR tube. The mixture was agitated with a
vortex mixer for 1 hour to reduce the disulfide bonds on the
antibody. 25% of DMSO was subsequently added to the reaction
mixtures. Compound 78 (5.0 equiv., 10 mM) was added to the reaction
mixtures and shaken overnight using a vortex mixer. All reactions
were carried out at room temperature. After reaction was over, the
mixture was loaded into the Millipore 4 mL centrifugal
ultrafiltration tube, and washed three times with PBS. The DAR of
the resultant CD59-C78 was 3.66 based on HIC-HPLC.
[0271] FIG. 6 shows the result of reduced SDS-PAGE for, a) naked
antibody, b) CD59-C78.
[0272] FIG. 7 shows an HIC-HPLC chromatogram of CD59-C78.
Example II-4 Preparation of HER2-C78 Conjugate
[0273] DTPA (1 mM) and 5.0 equiv. of TCEP (10 mM) were added to
anti-Her2 (10 mg/mL) in a PCR tube. The mixture was agitated with a
vortex mixer for 1 hour to reduce the disulfide bonds on the
antibody. 25% of DMSO was subsequently added to the reaction
mixtures. Compound 75 (5.0 equiv., 10 mM) was added to the reaction
mixtures and shaken overnight using a vortex mixer. All reactions
were carried out at room temperature. After reaction was over, the
mixture was loaded into the Millipore 4 mL centrifugal
ultrafiltration tube, and washed three times with PBS.
Stability Study of HER2-C78 Conjugate
[0274] To determine the degradation rates of ADCs of covalent thiol
conjugation vs. regular thiol conjugation, three ADC samples
prepared by above procedures for the ELISA were used for stability
study. ELISA implementation plan as follow: Firstly, Her2-ECD (100
ng) were coated on each well of a 96-well plate; Secondly, human
peripheral blood treated with Anti-Her2-Mc-VC-PAB-MMAE (of regular
thiol conjugation), Anti-Her2-Di-Mc-VC-PAB-MMAE (bi-maleimide-thiol
conjugation) and Anti-Her2-PY-QJ2-ADC (Her2-C78, of covalent-thiol
conjugation of this invention) were incubated in the coated well to
allow binding to Her-2 ECD. At last, the amount of anti-her2 ADCs
bound to Her2-ECD antigen is measured by binding anti-human IgG
conjugated with a streptavidin/horseradish peroxidase, which then
catalyzes the conversion of the chromogenic substrate
o-phenylenediamine (OPD) into a colored product. The results show
that during 3 days of incubation with peripheral bloods, the
degradation rate of ADC with the regular thiol conjugation was
26.7%, that of 2nd ADC with bi-maleimide-thiol conjugation was
18.9% and that of 3rd ADC of this invention was 5.1%. The
degradation rate of ADC with the covalent linker of this invention
is the lowest among the three ADCs as shown in Table 3 below.
TABLE-US-00003 TABLE 3 Summary of anti-Her2 drug conjugate
degradation rates in human blood Compounds Degradation Rate (3
days) Anti-Her2-Mc-VC-PAB-MMAE 26.7% Anti-Her2-Di-Mc-VC-PAB-MMAE
18.9% Anti-Her2-PY-QJ2-ADC (Her2-C78) 5.1% Notes:
Anti-Her2-Mc-VC-PAB-MMAE was conventional thiol-conjugation (ref-1)
and Anti-Her2-Di-Mc-VC-PAB-MMAE was of covalent conjugation using
the structure and procedure described in the patent publication
(Ref-2). DAR values of both conjugates were comparable to that of
Her2-C78.
Example III--Activity of the Antibody Drug Conjugates
[0275] The antibody drug conjugates of the subject invention can be
used, in one embodiment, to specifically target tumor cells and
bind to tumor-associated antigens on the surface of the tumor
cells. The complex of ADC-antigen will enter the tumor cell via
endocytosis and releases its payload after the endosome of the
ADC-antigen complex fuses with lysosomes. The released payloads
inhibit the key functions of a tumor cell and/or even kill the
tumor cells. If the payloads releases on the cell surface or near
the tumor cells, the tumor cell can also be killed via a by-stander
manner.
[0276] Thus, the invention provides various protein-drug complexes
that inhibit the growth of tumor cells and/or their
differentiation. These compounds, armed with various kinds of
active payloads, can be used to treat neoplasm, autoimmune
disorders and inflammation, even the differentiations of a tumor or
stem cell.
Example III-1--Activity Against Tumors
[0277] In one embodiment, or the subject invention, antibody drug
conjugates (ADCs) combine the unique targeting of mAbs with the
cancer-killing ability of cytotoxic drugs and allow ADCs to be
powerful tumor-killing agents. Furthermore, the use of covalent
thiol-conjugation in accordance with the subject invention
increases the stability of the ADCs, limiting the decomposition of
the ADC in the blood circulation, thereby reducing its toxicity.
This example provides data showing treatment of human cancer, using
ADCs prepared as described above, in nude mice implanted human
breast cancer cells (MDA-MB-231).
[0278] To determine biological function of various ADC with
covalent or regular conjugates, anti-Her-2 mAb has been
thiol-conjugated with regular conjugation or covalent
thiol-conjugated with the same linker and cytotoxin. The nude mice
were implanted with a human cancer cell line (MDA-MB-231) and the
ADCs (0, 2.5, 5.0 and 10 mg/kg) were administered through
intraperitoneal injection when the tumor volume reached about 100
cubic millimeters. The tumor volumes were monitored every 3 days
and animals were sacrificed at the end of 21 days and the tumors
were dissected and weighed.
[0279] Table-4 below provides a comparison of efficacy between
regular ADC vs. covalent thiol-conjugation ADC administered into
nude mice implanted with human breast cancer cells.
TABLE-US-00004 TABLE 4 Covalent thiol-conjugated ADC Inhibits human
tumor cell growth in nude mice study Treatments Treatment Time
Tumor volume in cubic mm (10 mg/kg) (Days) (% of Vehicle) Vehicle
21 1084 (100%).sup. Regular ADC 21 536 (49.5%) Covalent-ADC 21 450
(41.6%)
Example III-2-Comparison of Antitumor Effects of Antibody Drug
Conjugates with Different Structures on Subcutaneous Xenografts of
Human Gastric Cancer in N87 Nude Mice
[0280] 1. The Sources of Samples and Reagents
[0281] Anti-Her2-Mc-VC-PAB-MMAE at a concentration of 2.874 mg/mL
with batch number: 20171009 is from RC Biotechnologies, Inc.
[0282] Anti-Her2-C75 at a concentration of 9.885 mg/mL with a batch
number: 20171009 is from RC Biotechnologies, Inc.
[0283] RPMI Medium 1640 (lx), Gibco, 500 mL/bottle, batch number:
1868807
[0284] Fetal bovine serum, EX Cell Bio, 500 mL/bottle, batch
number: 11E100
[0285] Matrigel matrix, BD, 10 mL/bottle (10 mg/mL), batch number:
354243
[0286] 2. The Sources of Animal and Cell Line
[0287] 4.about.5 weeks old, female BALB/c nude mice of SPF grade
were purchased from Changzhou Cavens Experimental Animal Co., Ltd.,
Animal Quality Certificate No. 201717799.
[0288] Human gastric cancer cell line N87 was purchased from
ATCC.
[0289] 3. Experimental Methods
[0290] BALB/c nude mice (purchased from Changzhou Cavince
Experimental Animal Co., Ltd.) were subcutaneously inoculated with
N87 cells (purchased from ATCC) for 2.times.10.sup.6 cells per
mouse under the right armpit near the back side to establish a
subcutaneous xenograft model. On the 11.sup.th day after
inoculation of the tumor cells, qualified tumor-bearing animals of
similar body weight were selected and randomly divided into 7
groups according to the tumor volume: the control group, the groups
of Anti-Her2-Mc-VC-PAB-MMAE of three doses (0.5 mg/kg, 1 mg/kg, and
2 mg/kg, intravenously (iv.)), and Anti-Her2-C75 of three doses
(0.5 mg/kg, 1 mg/kg, and 2 mg/kg, iv.). After grouping, drugs were
administered once a week for 3 times, i.e., Day 1 (on the day of
administration), Day 8 and D15 respectively. The animals in the
control group were given the same volume of normal saline by tail
vein injection. During the experiment, the animals were weighed
twice a week. Meanwhile, the animals were observed for death or
dying, the rupture of tumor nodules, the mental state of animals,
behavior activities, feces characteristics, the supply of feed and
drinking water, and so on. The long diameter and short diameter of
the tumor were measured with a vernier caliper and recorded, and
the tumor volume (TV) was calculated. The tumor diameter was
measured once before grouping, and twice a week after the first
dose.
[0291] 4. Experimental Results
[0292] As shown in FIGS. 8 and 9, during the experiment, the body
weight for the animals of each group gradually increased. No weight
loss due to toxic effects, and no significant abnormal changes in
other clinical signs were observed. Anti-Her2-Mc-VC-PAB-MMAE of 0.5
mg/kg less effectively inhibited tumor growth, and doses of 1 mg/kg
and 2 mg/kg significantly inhibited tumor growth, wherein a dose of
2 mg/kg showed a better effect. Anti-Her2-C75 of each dose
significantly inhibited tumor growth. Anti-Her2-C75 of 0.5 mg/kg
and 1 mg/kg had equivalent inhibition on tumor growth.
Anti-Her2-C75 of 2 mg/kg has stronger inhibition on tumor growth,
superior to Anti-Her2-Mc-VC-PAB-MMAE of an equal dose.
REFERENCES
[0293] 1. Yao Xuejing, Jing Jiang, Xin Wang, Changjiang Huang Dong
Li, Kuan Xie, Qiaoyu Xu, Hongwen Li, Zhuanglin Li, Liguang Lou and
Jianmin Fang, 2015 "A novel humanized anti-her2 antibody conjugated
with MMAE exerts potent anti-tumor activity," Breast Cancer
Research & Treatment, 153(1):123-133. [0294] 2. An Deqiang et
al., 2014 Jul. 23 CN103933575A
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